Catalytically active recombinant memapsin and methods of use thereof

ABSTRACT

Methods for the production of purified, catalytically active, recombinant memapsin 2 have been developed. The substrate and subsite specificity of the catalytically active enzyme have been determined. The substrate and subsite specificity information was used to design substrate analogs of the natural memapsin 2 substrate that can inhibit the function of memapsin 2. The substrate analogs are based on peptide sequences, shown to be related to the natural peptide substrates for memapsin 2. The substrate analogs contain at least one analog of an amide bond which is not capable of being cleaved by memapsin 2. Processes for the synthesis of two substrate analogues including isosteres at the sites of the critical amino acid residues were developed and the substrate analogues, OMR99-1 and OM99-2, were synthesized. OM99-2 is based on an octapeptide Glu-Val-Asn-Leu-Ala-Ala-Glu-Phe (SEQ ID NO:28) with the Leu-Ala peptide bond substituted by a transition-state isostere hydroxyethylene group (FIG.  1 ). The inhibition constant of OM99-2 is 1.6×10 −9  M against recombinant pro-memapsin 2. Crystallography of memapsin 2 bond to this inhibitor was used to determine the three dimensional structure of the protein, as well as the importance of the various residues in binding. This information can be used by those skilled in the art to design new inhibitors, using commercially available software programs and techniques familiar to those in organic chemistry and enzymology, to design new inhibitors to memapsin 2, useful in diagnostics and for the treatment and/or prevention of Alzheimer&#39;s disease.

This application is a continuation of application Ser. No. 11/763,342,filed Jun. 14, 2007, which is a continuation of application Ser. No.10/773,754, filed Feb. 6, 2004, which is a continuation of applicationSer. No. 09/845,226, filed Apr. 30, 2001 (now abandoned), which is adivisional of application Ser. No. 09/603,713, filed Jun. 27, 2000 (nowabandoned), by Gerald Koelsch, Jordan J. N. Tang, Lin Hong, and Arun K.Ghosh, which claims priority to provisional application Nos. 60/141,363filed Jun. 28, 1999 by Lin, et al., 60/168,060, filed Nov. 30, 1999 byLin, et al., 60/177,836 filed Jan. 25, 2000 by Lin, et al., 60/178,368,filed Jan. 27, 2000 by Lin, et al., and 60/210,292, filed Jun. 8, 2000by Lin Hong, et al., the teachings of which are incorporated byreference herein.

BACKGROUND OF THE INVENTION

This invention is in the area of the design and synthesis of specificinhibitors of the aspartic protease Memapsin 2 (beta-secretase) whichare useful in the treatment and/or prevention of Alzheimer's Disease.

Alzheimer's disease (AD) is a degenerative disorder of the brain firstdescribed by Alios Alzheimer in 1907 after examining one of his patientswho suffered drastic reduction in cognitive abilities and hadgeneralized dementia (The early story of Alzheimer's Disease, edited byBick et al. (Raven Press, New York 1987)). It is the leading cause ofdementia in elderly persons. AD patients have increased problems withmemory loss and intellectual functions which progress to the point wherethey cannot function as normal individuals. With the loss ofintellectual skills the patients exhibit personality changes, sociallyinappropriate actions and schizophrenia (A Guide to the Understanding ofAlzheimer's Disease and Related Disorders, edited by Jorm (New YorkUniversity Press, New York 1987). AD is devastating for both victims andtheir families, for there is no effective palliative or preventivetreatment for the inevitable neurodegeneration.

AD is associated with neuritic plaques measuring up to 200 μm indiameter in the cortex, hippocampus, subiculum, hippocampal gyrus, andamygdala. One of the principal constituents of neuritic plaques isamyloid, which is stained by Congo Red (Fisher (1983); Kelly Microbiol.Sci. 1(9):214-219 (1984)). Amyloid plaques stained by Congo Red areextracellular, pink or rust-colored in bright field, and birefringent inpolarized light. The plaques are composed of polypeptide fibrils and areoften present around blood vessels, reducing blood supply to variousneurons in the brain.

Various factors such as genetic predisposition, infectious agents,toxins, metals, and head trauma have all been suggested as possiblemechanisms of AD neuropathy. Available evidence strongly indicates thatthere are distinct types of genetic predispositions for AD. First,molecular analysis has provided evidence for mutations in the amyloidprecursor protein (APP) gene in certain AD-stricken families (Goate etal. Nature 349:704-706 (1991); Murrell et al. Science 254:97-99 (1991);Chartier-Harlin et al. Nature 353:844-846 (1991); Mullan et al., NatureGenet. 1:345-347 (1992)). Additional genes for dominant forms of earlyonset AD reside on chromosome 14 and chromosome 1 (Rogaev et al., Nature376:775-778 (1995); Levy-Lahad et al., Science 269:973-977 (1995);Sherrington et al., Nature 375:754-760 (1995)). Another loci associatedwith AD resides on chromosome 19 and encodes a variant form ofapolipoprotein E (Corder, Science 261:921-923 (1993)).

Amyloid plaques are abundantly present in AD patients and in Down'sSyndrome individuals surviving to the age of 40. The overexpression ofAPP in Down's Syndrome is recognized as a possible cause of thedevelopment of AD in Down's patients over thirty years of age (Rumble etal., New England J. Med. 320:1446-1452 (1989); Main et al., Neurobiol.Aging 10:397-399 (1989)). The plaques are also present in the normalaging brain, although at a lower number. These plaques are made upprimarily of the amyloid β peptide (Aβ; sometimes also referred to inthe literature as β-amyloid peptide or (3 peptide) (Glenner and Wong,Biochem. Biophys. Res. Comm. 120:885-890 (1984)), which is also theprimary protein constituent in cerebrovascular amyloid deposits. Theamyloid is a filamentous material that is arranged in beta-pleatedsheets. Aβ is a hydrophobic peptide comprising up to 43 amino acids.

The determination of its amino acid sequence led to the cloning of theAPP cDNA (Kang et al., Nature 325:733-735 (1987); Goldgaber et al.,Science 235:877-880 (1987); Robakis et al., Proc. Natl. Acad. Sci.84:4190-4194 (1987); Tanzi et al., Nature 331:528-530 (1988)) andgenomic APP DNA (Lemaire et al., Nucl. Acids Res. 17:517-522 (1989);Yoshikai et al., Gene 87, 257-263 (1990)). A number of forms of APP cDNAhave been identified, including the three most abundant forms, APP695,APP751, and APP770. These forms arise from a single precursor RNA byalternate splicing. The gene spans more than 175 kb with 18 exons(Yoshikai et al. (1990)). APP contains an extracellular domain, atransmembrane region and a cytoplasmic domain. Aβ consists of up to 28amino acids just outside the hydrophobic transmembrane domain and up to15 residues of this transmembrane domain. Aβ is normally found in brainand other tissues such as heart, kidney and spleen. However, Aβ depositsare usually found in abundance only in the brain.

Van Broeckhaven et al., Science 248:1120-1122 (1990), have demonstratedthat the APP gene is tightly linked to hereditary cerebral hemorrhagewith amyloidosis (HCHWA-D) in two Dutch families. This was confirmed bythe finding of a point mutation in the APP coding region in two Dutchpatients (Levy et al., Science 248:1124-1128 (1990)). The mutationsubstituted a glutamine for glutamic acid at position 22 of the Aβ(position 618 of APP695, or position 693 of APP770). In addition,certain families are genetically predisposed to Alzheimer's disease, acondition referred to as familial Alzheimer's disease (FAD), throughmutations resulting in an amino acid replacement at position 717 of thefull length protein (Goate et al. (1991); Murrell et al. (1991);Chartier-Harlin et al. (1991)). These mutations co-segregate with thedisease within the families and are absent in families with late-onsetAD. This mutation at amino acid 717 increases the production of theAβ₁₋₄₂ form of Aβ from APP (Suzuki et al., Science 264:1336-1340(1994)). Another mutant form contains a change in amino acids atpositions 670 and 671 of the full length protein (Mullan et al. (1992)).This mutation to amino acids 670 and 671 increases the production oftotal Aβ from APP (Citron et al., Nature 360:622-674 (1992)).

APP is processed in vivo at three sites. The evidence suggests thatcleavage at the β-secretase site by a membrane associatedmetalloprotease is a physiological event. This site is located in APP 12residues away from the lumenal surface of the plasma membrane. Cleavageof the β-secretase site (28 residues from the plasma membrane's lumenalsurface) and the β-secretase site (in the transmembrane region) resultsin the 40/42-residue β-amyloid peptide (A β), whose elevated productionand accumulation in the brain are the central events in the pathogenesisof Alzheimer's disease (for review, see Selkoe, D. J. Nature 399:23-31(1999)). Presenilin 1, another membrane protein found in human brain,controls the hydrolysis at the APP (β-secretase site and has beenpostulated to be itself the responsible protease (Wolfe, M. S. et al.,Nature 398:513-517 (1999)). Presenilin 1 is expressed as a single chainmolecule and its processing by a protease, presenilinase, is required toprevent it from rapid degradation (Thinakaran, G. et al., Neuron17:181-190 (1996) and Podlisny, M. B., et al., Neurobiol. Dis. 3:325-37(1997)). The identity of presenilinase is unknown. The in vivoprocessing of the β-secretase site is thought to be the rate-limitingstep in A β-production (Siniha, S. & Lieberburg, I., Proc. Natl. Acad.Sci., USA, 96:11049-11053 (1999)), and is therefore a strong therapeutictarget.

The design of inhibitors effective in decreasing amyeloid plaqueformation is dependent on the identification of the critical enzyme(s)in the cleavage of APP to yield the 42 amino acid peptide, the Aβ₁₋₄₂form of Aβ. Although several enzymes have been identified, it has notbeen possible to produce active enzyme. Without active enzyme, onecannot confirm the substrate specificity, determine the subsitespecificity, nor determine the kinetics or critical active siteresidues, all of which are essential for the design of inhibitors.

Memapsin 2 has been shown to be beta-secretase, a key protease involvedin the production in human brain of beta-amyloid peptide frombeta-amyloid precursor protein (for review, see Selkoe, D. J. Nature399:23-31 (1999)). It is now generally accepted that the accumulation ofbeta-amyloid peptide in human brain is a major cause for the Alzheimer'sdisease. Inhibitors specifically designed for human memapsin 2 shouldinhibit or decrease the formation of beta-amyloid peptide and theprogression of the Alzheimer's disease.

Memapsin 2 belongs to the aspartic protease family. It is homologous inamino acid sequence to other eukaryotic aspartic proteases and containsmotifs specific to that family. These structural similarities predictthat memapsin 2 and other eukaryotic aspartic proteases share commoncatalytic mechanism Davies, D. R., Annu. Rev. Biophys. Chem. 19, 189(1990). The most successful inhibitors for aspartic proteases are mimicsof the transition state of these enzymes. These inhibitors havesubstrate-like structure with the cleaved planar peptide bond betweenthe carbonyl carbon and the amide nitrogen replaced by two tetrahedralatoms, such as hydroxyethylene [—CH(OH)—CH₂—], which was originallydiscovered in the structure of pepstatin (Marciniszyn et al., 1976).

However, for clinical use, it is preferable to have small moleculeinhibitors which will pass through the blood brain barrier and which canbe readily synthesized. It is also desirable that the inhibitors arerelatively inexpensive to manufacture and that they can be administeredorally. Screening of thousands of compounds for these properties wouldrequire an enormous effort. To rationally design memapsin 2 inhibitorsfor treating Alzheimer's disease, it will be important to know thethree-dimensional structure of memapsin 2, especially the binding modeof an inhibitor in the active site of this protease.

It is therefore an object of the present invention to provide purified,recombinant, and active memapsin 2, as well as its substrate and subsitespecificity and critical active site residues.

It is a further object of the present invention to provide compositionsand methods for synthesis of inhibitors of memapsin 2.

It is a still further object of the present invention to providecompositions that interact with memapsin 2 or its substrate to inhibitcleavage by the memapsin 2 which can cross the blood brain barrier(BBB).

It is therefore an object of the present invention to provide means forrational design and screening of compounds for inhibition of mamapsin 2.

SUMMARY OF THE INVENTION

Methods for the production of purified, catalytically active,recombinant memapsin 2 have been developed. The substrate and subsitespecificity of the catalytically active enzyme have been determined. Theactive enzyme and assays for catalytic activity are useful in screeninglibraries for inhibitors of the enzyme.

The substrate and subsite specificity information was used to designsubstrate analogs of the natural memapsin 2 substrate that can inhibitthe function of memapsin 2. The substrate analogs are based on peptidesequences, shown to be related to the natural peptide substrates formemapsin 2. The substrate analogs contain at least one analog of anamide (peptide) bond which is not capable of being cleaved by memapsin2. Processes for the synthesis of two substrate analogues includingisosteres at the sites of the critical amino acid residues weredeveloped and the substrate analogues, OMR99-1 and OM99-2, weresynthesized. OM99-2 is based on an octapeptideGlu-Val-Asn-Leu-Ala-Ala-Glu-Phe (SEQ ID NO:28) with the Leu-Ala peptidebond substituted by a transition-state isostere hydroxyethylene group.The inhibition constant of OM99-2 is 1.6×10⁻⁹ M against recombinantpro-memapsin 2. Crystallography of memapsin 2 bound to this inhibitorwas used to determine the three dimensional structure of the protein, aswell as the importance of the various residues in binding.

This information can be used by those skilled in the art to design newinhibitors, using commercially available software programs andtechniques familiar to those in organic chemistry and enzymology, todesign new inhibitors. For example, the side chains of the inhibitorsmay be modified to produce stronger interactions (through hydrogenbonding, hydrophobic interaction, charge interaction and/or van der Waalinteraction) in order to increase inhibition potency. Based on this typeof information, the residues with minor interactions may be eliminatedfrom the new inhibitor design to decrease the molecular weight of theinhibitor. The side chains with no structural hindrance from the enzymemay be cross-linked to lock in the effective inhibitor conformation.This type of structure also enables the design of peptide surrogateswhich may effectively fill the binding sites of memapsin 2 yet producebetter pharmaceutical properties.

The examples demonstrate the production of catalytically active enzyme,design and synthesis of inhibitors, and how the crystal structure wasobtained. The examples thereby demonstrate how the methods and materialsdescribed herein can be used to screen libraries of compounds for otherinhibitors, as well as for design of inhibitors. These inhibitors areuseful in the prevention and/or treatment of Alzheimer's disease asmediated by the action of the beta secretase memapsin 2, in cleavingAPP.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the plasmid construct of vector pET-11a-memapsin 2-T1 andpET-11a-memapsin 2 T2. The T7 promoter, amino acid sequence from thevector (T7 protein) (SEQ ID NO:3), and the beginning and ending of thememapsin 2 T1 and T2 construct are shown (SEQ ID NOS:32-34). Constructpromemapsin 2-T1 was used in the preparation of protein forcrystallization and includes residues 1v-15v (SEQ ID NO:43) which arederived from vector pET-11a. Residues 1p-48p (SEQ ID NO:44) are putativepro-peptide. Residues 1-393 (SEQ ID NO:45) correspond to the matureprotease domain and C-terminal extension. The residue numbering ofmemapsin 2 starts at the aligned N-terminal position of pepsin.

FIG. 2A is a graph of the initial rate of hydrolysis of syntheticpeptide swAPP (see Table 1) by M2_(pd) at different pH. FIG. 2B is agraph of the relative k_(cat)/K_(m) values for steady-state kinetic ofhydrolysis of peptide substrates by M2_(pd).

FIGS. 3A and 3B are the chemical structures of memapsin 2 inhibitors,OM99-1 (SEQ ID NO:27) and OM99-2 (SEQ ID NO:35).

FIG. 4A is a graph of the inhibition of recombinant memapsin 2 byOM99-1. FIG. 4B is a graph of the inhibition of recombinant memapsin 2by OM99-2.

FIGS. 5A-E are photographs of crystals of recombinant memapsin 2-OM99-2complex.

FIG. 6 is a stereo view of crystal structure of memapsin 2 proteasedomain with bound 0M99-2. The polypeptide backbone of memapsin 2 isshown as a ribbon diagram. The N-lobe and C-lobe are labeled “Blue” and“Yellow,” respectively, except the insertion loops (designated A to G,see FIG. 6) in the C-lobe are labeled “Magenta” and the C-terminalextension is labeled “Green.” The inhibitor bound between the lobes islabeled “Red.”

FIG. 7 is a stereo view of comparison of the three-dimensionalstructures of memapsin 2 and pepsin. The molecular surface of the formeris significantly larger by the insertion of surface loops and helix andthe C-terminal extension. Chain tracing of human memapsin 2 is labeled“Dark Blue” and is labeled “Grey” for human pepsin. The balls labeled as“Light Blue” represent identical residues which are topologicallyequivalent. The disulfide bonds are labeled “Red” for memapsin 2 and“Orange” for pepsin. The C-terminal extension is labeled “Green.”

FIG. 8 is a schematic presentation of interaction between OM99-2 (SEQ IDNO:35) and memapsin 2 protease domain. The S₃′ and S₄′ subsites are notdefined.

FIG. 9 is a stereo presentation of interactions between inhibitor OM99-2(labeled “Orange”) and memapsin 2 (labeled “Light Blue”). Nitrogen andoxygen atoms are labeled “Blue” and “Red”, respectively. Hydrogen bondsare indicated as dotted lines. Memapsin 2 residues which comprise thebinding subsites are included. Residues P₄, P₃, P₂, P₁ and R_(1′)(defined in FIG. 8) of OM99-2 are in an extended conformation. Inhibitorchain turns at residue P2′ which makes a distinct kink at this position.The backbone of P_(3′) and P_(4′) directs the inhibitor to exit theactive site.

FIG. 10 are schematics of the cross linking between P₃ Val and P₁ Leuside chains in the design of new inhibitors for memapsin 2 based on thecurrent crystal structure. R and R′ at positions P₂ and P₁′ indicateamino acid side chains. Other structural elements of inhibitor areomitted for clarity.

FIG. 11 are schematics of the cross linking between P₄ Glu and P₂ Asnside chains in the design of new inhibitors for memapsin 2 based on thecurrent crystal structure. R at position P₃ indicates amino acid sidechain. Other structural elements of inhibitor are omitted for clarity.

FIG. 12 is a schematic of the design for the side chain at the P₁′subsite for the new memapsin 2 inhibitors based on the current crystalstructure. Arrows indicate possible interactions between memapsin 2 andinhibitor. Other structural elements of inhibitor are omitted forclarity.

FIG. 13 is a schematic of the design of two six-membered rings in theinhibitor structure by the addition of atoms A and B. The ring formationinvolves the P₁-Leu side chain the peptide backbone near P₁, P₂, and P₃.The new bonds are in dotted lines. A methyl group can be added to thebeta-carbon of P₁-Leu. Other structural elements of inhibitor areomitted for clarity.

DETAILED DESCRIPTION OF THE INVENTION I. Preparation of CatalyticallyActive Recombinant Memapsin 2

Cloning and Expression of Memapsin 2

Memapsin 2 was cloned and the nucleotide (SEQ ID NO. 1) and predictedamino acid (SEQ ID NO. 2) sequences were determined, as described inExample 1. The cDNA was assembled from the fragments. The nucleotide andthe deduced protein sequence are shown in SEQ ID NOs. 1 and 2,respectively. The protein is the same as the aspartic proteinase 2(ASP2) described in EP 0 855 444 A by SmithKline BeechamPharmaceuticals, (published Jul. 29, 1998), and later reported by Sinha,et al., Nature 402, 537-540 (December 1999) and Vassar, et al., Science286, 735-741 (22 Oct. 1999).

Pro-memapsin 2 is homologous to other human aspartic proteases. Based onthe alignments, Pro-memapsin 2 contains a pro region, an asparticprotease region, and a trans-membrane region near the C-terminus. TheC-terminal domain is over 80 residues long. The active enzyme ismemapsin 2 and its pro-enzyme is pro-memapsin 2.

Refolding Catalytically Active Enzyme

In order to determine the substrate specificity and to designinhibitors, it is necessary to express catalytically active recombinantenzyme. No other known proteases contain a transmembrane domain. Thepresence of transmembrane domains makes the recombinant expression ofthese proteins less predictable and more difficult. The transmembraneregion often needs to be removed so that secretion of the protein cantake place. However, the removal of the transmembrane region can oftenalter the structure and/or function of the protein.

The starting assumption was that the region of memapsin 2 that ishomologous with other aspartic proteases would independently fold in theabsence of the transmembrane domain, and would retain protease activityin the absence of the C-terminal transmembrane region. The transmembraneregion appears to serve as a membrane anchor. Since the active site isnot in the transmembrane region and activity does not require membraneanchoring, memapsin 2 was expressed in E. coli in two different lengths,both without the transmembrane region, and purified, as described inExample 3. The procedures for the culture of transfected bacteria,induction of synthesis of recombinant proteins and the recovery andwashing of inclusion bodies containing recombinant proteins areessentially as described by Lin et al., (1994). Refolding was not asimple matter, however. Two different refolding methods both producedsatisfactory results. In both methods, the protein was dissolved in astrong denaturing/reducing solution such as 8 M urea/100 mMbeta-mercaptoethanol. The rate at which the protein was refolded, and inwhat solution, was critical to activity. In one method, the protein isdissolved into 8 M urea/100 mM beta-mercaptoethanol then rapidly dilutedinto 20 volumes of 20 mM-Tris, pH 9.0, which is then slowly adjusted topH 8 with 1 M HCl. The refolding solution was then kept at 4° C. for 24to 48 hours before proceeding with purification. In the second method,an equal volume of 20 mM Tris, 0.5 mM oxidized/1.25 mM reducedglutathione, pH 9.0 is added to rapidly stirred pro-memapsin 2 in 8 Murea/10 mM beta-mercaptoethanol. The process is repeated three moretimes with 1 hour intervals. The resulting solution is then dialyzedagainst sufficient volume of 20 mM Tris base so that the final ureaconcentration is 0.4 M. The pH of the solution is then slowly adjustedto 8.0 with 1 M HCl.

The refolded protein is then further purified by column chromatography,based on molecular weight exclusion, and/or elution using a saltgradient, and analyzed by SDS-PAGE analysis under reduced andnon-reduced conditions.

II. Substrate Specificity, and Enzyme Kinetics of Memapsin 2

Substrate Specificity

The tissue distribution of the memapsin 2 was determined, as describedin Example 2. The presence of memapsin 2 (M2) in the brain indicatedthat it might hydrolyze the β-amyloid precursor protein (APP). Asdescribed below, detailed enzymatic and cellular studies demonstratedthat M2 fits all the criteria of the β-secretase.

The M2 three-dimensional structure modeled as a type I integral membraneprotein. The model suggested that its globular protease unit canhydrolyze a membrane anchored polypeptide at a distance range of 20-30residues from the membrane surface. As a transmembrane protein of thebrain, APP is a potential substrate and its beta-secretase site, locatedabout 28 residues from the plasma membrane surface, is within in therange for M2 proteolysis.

A synthetic peptide derived from this site (SEVKM/DAEFR) (SEQ ID NO:4)was hydrolyzed by M2_(pd) at the beta-secretase site (marked by /). Asecond peptide (SEVNL/DAEFR) (SEQ ID NO:5) derived from the APPbeta-secretase site and containing the ‘Swedish mutation’ (Mullan, M. etal., Nature Genet. 2:340-342 (1992)), known to elevate the level ofalpha-beta production in cells (Citron, M. et al., Nature 260:672-674(1992)), was hydrolyzed by M2_(pd) with much higher catalyticefficiency. Both substrates were optimally cleaved at pH 4.0. A peptidederived from the processing site of presenilin 1 (SVNM/AEGD) (SEQ IDNO:6) was also cleaved by M2_(pd) with less efficient kineticparameters. A peptide derived from the APP gamma-secretase site(KGGVVIATVIVK) (SEQ ID NO:7) was not cleaved by M2_(pd). Pepstatin Ainhibited M2_(pd) poorly (IC₅₀ approximately approximately 0.3 mM). Thekinetic parameters indicate that both presenilin 1 (k_(cat), 0.67 s⁻¹;K_(m), 15.2 mM; k_(cat)/K_(m), 43.8 s⁻¹M⁻¹) and native APP peptides(k_(cat)/K_(m), 39.9 s⁻¹M⁻¹) are not as good substrates as the SwedishAPP peptide (k_(cat), 2.45 s⁻¹, K_(m), 1 mM; k_(cat)/K_(m), 2450s⁻¹M⁻¹).

To determine if M2 possesses an APP beta-secretase function in mammaliancells, memapsin 2 was transiently expressed in HeLa cells (Lin, X., etal., FASEB J. 7:1070-1080 (1993)), metabolically pulse-labeled with³⁵S-Met, then immunoprecipitated with anti-APP antibodies forvisualization of APP-generated fragments after SDS-polyacrylamideelectrophoresis and imaging. SDS-PAGE patterns of immuno-precipitatedAPP Nβ-fragment (97 kD band) from the conditioned media (2 h) ofpulse-chase experiments showed that APP was cleaved by M2. Controlstransfected with APP alone and co-transfected with APP and M2 withBafilomycin A1 added were performed. SDS-PAGE patterns of APPβC-fragment (12 kD) were immunoprecipitated from the conditioned mediaof the same experiment as discussed above. Controls transfected with APPalone; co-transfected with APP and M2; co-transfected with APP and M2with Bafilomycin A1; transfections of Swedish APP; and co-transfectionsof Swedish APP and M2 were performed. SDS-PAGE gels were also run ofimmuno-precipitated M2 (70 kD), M2 transfected cells; untransfected HeLacells after long time film exposure; and endogenous M2 from HEK 293cells. SDS-PAGE patterns of APP fragments (100 kD betaN-fragment and 95kD betaN-fragment) recovered from conditioned media afterimmuno-precipitation using antibodies specific for different APP regionsindicated that memapsin 2 cleaved APP.

Cells expressing both APP and M2 produced the 97 kD APP beta N-fragment(from the N-terminus to the beta-secretase site) in the conditionedmedia and the 12 kD betaC-fragment (from the beta-secretase site to theC-terminus) in the cell lystate. Controls transfected with APP aloneproduced little detectable betaN-fragment and no beta C-fragment.Bafilomycin A1, which is known to raise the intra-vesicle pH oflysosomes/endosomes and has been shown to inhibit APP cleavage bybeta-secretase (Knops, J. et al., J. Biol. Chem. 270:2419-2422 (1995)),abolished the production of both APP fragments beta N- and beta C- inco-transfected cells. Cells transfected with Swedish APP alone did notproduce the beta C-fragment band in the cell lysate but theco-transfection of Swedish APP and M2 did. This Swedish beta C-fragmentband is more intense than that of wild-type APP. A 97-kD beta N-band isalso seen in the conditioned media but is about equal intensity as thewild-type APP transfection.

These results indicate that M2 processes the beta-secretase site of APPin acidic compartments such as the endosomes. To establish theexpression of transfected M2 gene, the pulse-labeled cells were lysedand immuno-precipitated by anti-M2 antibodies. A 70 kD M2 band was seenin cells transfected with M2 gene, which has the same mobility as themajor band from HEK 293 cells known to express beta-secretase (Citron,M. et al., Nature 260:672-674 (1992)). A very faint band of M2 is alsoseen, after a long film exposure, in untransfected HeLa cells,indicating a verb low level of endogenous M2, which is insufficient toproduce betaN- or betaC-fragments without M2 transfection. Antibodyalpha-beta₁₋₁₇, which specifically recognizes residues 1-17 inalpha-beta peptide, was used to confirm the correct beta-secretase sitecleavage. In cells transfected with APP and M2, both beta N- and betaN-fragments are visible using an antibody recognizing the N-terminalregion of APP present in both fragments. Antibody Abeta₁₋₁₇ recognizethe beta N-fragment produced by endogenous beta-secretase in theuntransfected cells. This antibody was, however, unable to recognize thebetaN-fragment known to be present in cells co-transfected with APP andM2. These observations confined that betaN-fragment is the product ofbeta-secretase site cut by M2, which abolished the recognition epitopeof alpha-beta₁₋₁₇.

The processing of APP by M2 predicts the intracellular colocalization ofthe two proteins. HeLa cells co-expressing APP and M2 were stained withantibodies directed toward APP and M2 and visualized simultaneously byCSLM using a 100× objective. Areas of colocalization appeared in yellow.

Immunodetection observed by confocal microscopy of both APP and M2revealed their colocalization in the superimposed scans. Thedistribution of both proteins is consistent with their residence inlysosomal/endosomal compartments.

In specificity studies, it was found that M2_(pd) cleaved its propeptide (2 sites) and the protease portion (2 sites) during a 16 hincubation after activation (Table 1). Besides the three peptidesdiscussed above, M2_(pd) also cleaved oxidized bovine insulin B chainand a synthetic peptide Nch. Native proteins were not cleaved byM2_(pd).

The data indicate that human M2 fulfills all the criteria of abeta-secretase which cleaves the beta-amyloid precursor protein (APP):(a) M2 and APP are both membrane proteins present in human brain andco-localize in mammalian cells, (b) M2 specifically cleaves thebeta-secretase site of synthetic peptides and of APP in cells, (c) M2preferentially cleaves the beta-secretase site from the Swedish over thewild-type APP, and (d) the acidic pH optimum for M2 activity andbafilomycin A1 inhibition of APP processing by M2 in the cells areconsistent with the previous observations that beta-secretase cleavageoccurs in acidic vesicles (Knops, J., et al., J. Biol. Chem.270:2419-2422 (1995)). The spontaneous appearance of activity ofrecombinant pro-M2 in an acidic solution suggests that, intracellularly,this zymogen can by itself generate activity in an acidic vesicle likean endosome.

II. Design and Synthesis of Inhibitors

Design of Substrate Analogs for Memapsin 2.

The five human aspartic proteases have homologous amino acid sequencesand have similar three-dimensional structures. There are two asparticresidues in the active site and each residue is found within thesignature aspartic protease sequence motif, Asp-Thr/Ser-Gly- (SEQ IDNO:8). There are generally two homologous domains within an asparticprotease and the substrate binding site is positioned between these twodomains, based on the three-dimensional structures. The substratebinding sites of aspartic proteases generally recognize eight amino acidresidues. There are generally four residues on each side of the amidebond which is cleaved by the aspartic protease.

Typically the side chains of each amino acid are involved in thespecificity of the substrate/aspartic protease interaction. The sidechain of each substrate residue is recognized by regions of the enzymewhich are collectively called sub-sites. The generally acceptednomenclature for the protease sub-sites and their correspondingsubstrate residues are shown below, where the double slash representsthe position of bond cleavage.

Protease sub-sites S4 S3 S2 S1 S1′ S2′ S3′ S4′ Substrate residues P4 P3P2 P1 // P1′ P2′ P3′ P4′

While there is a general motif for aspartic protease substraterecognition, each protease has a very different substrate specificityand breadth of specificity. Once the specificity of an aspartic proteaseis known, inhibitors can be designed based on that specificity, whichinteract with the aspartic protease in a way that prevents naturalsubstrate from being efficiently cleaved. Some aspartic proteases havespecificities which can accommodate many different residues in each ofthe sub-sites for successful hydrolysis. Pepsin and cathepsin D havethis type of specificity and are said to have “broad” substratespecificity. When only a very few residues can be recognized at asub-site, such as in renin, the aspartic protease is said to have astringent or narrow specificity.

The information on the specificity of an aspartic protease can be usedto design specific inhibitors in which the preferred residues are placedat specific sub-sites and the cleaved peptide bond is replaced by alanalog of the transition-state. These analogs are called transitionstate isosteres. Aspartic proteases cleave amide bonds by a hydrolyticmechanism. This reaction mechanism involves the attack by a hydroxideion on the β-carbon of the amino acid. Protonation must occur at theother atom attached to the β-carbon through the bond that is to becleaved. If the β-carbon is insufficiently electrophilic or the atomattached to the bond to be cleaved is insufficiently nucleophilic thebond will not be cleaved by a hydrolytic mechanism. Analogs exist whichdo not mimic the transition state but which are non-hydrolyzable, buttransition state isosteres mimic the transition state specifically andare non-hydrolyzable.

Transition state theory indicates that it is the transition stateintermediate of the reaction which the enzyme catalyzes for which theenzyme has its highest affinity. It is the transition state structure,not the ground state structure, of the substrate which will haze thehighest affinity for its given enzyme. The transition state for thehydrolysis of an amide bond is tetrahedral while the ground statestructure is planar. A typical transition-state isostere of asparticprotease is —CH(OH)—CH₂—, as was first discovered in pepstatin byMarciniszyn et al. (1976). The transition-state analogue principles havebeen successfully applied to inhibitor drugs for human immunodeficiencyvirus protease, an aspartic protease. Many of these are currently inclinical use. Information on the structure, specificity, and types ofinhibitors can be found in Tang, Acid Proteases, Structure, Function andBiology, Adv. in Exptl. Med. Biol. vol. 95 (Plenum Press, NY 1977);Kostka, Aspartic Proteinases and their Inhibitors (Walter de Gruyter,Berlin 1985); Dunn, Structure and Functions of the Aspartic Proteinases,Adv. in Exptl. Med. Biol. 306 (Plenum Press, NY 1991); Takahashi,Aspartic Proteases, Structure, Function, Biology, BiomedicalImplications, Adv. in Exptl. Med. Biol. 362 (Plenum Press, NY 1995); andJames, Aspartic Proteinases, Retroviral and Cellular Enzymes, Adv. inExptl. Med. Biol. 436 (Plenum Press, NY 1998)).

Substrate analog compositions are generally of the general formulaX-L₄-P₄-L₃-P₃-L₂-P₂-L₁-P₁-L₀-P₁′-L₁′-P₂′-L₂′-P₃′-L₃′-P₄′L₄′-Y. Thesubstrate analog compositions are analogs of small peptide molecules.Their basic structure is derived from peptide sequences that weredetermined through structure/function studies. It is understood thatpositions represented by P_(x) represent the substrate specificityposition relative to the cleavage site which is represented by an -L₀-.The positions of the compositions represented by L_(x) represent thelinking regions between each substrate specificity position, P_(x).

In a natural substrate for memapsin 2, a P_(x)-L_(x) pair wouldrepresent a single amino acid of the peptide which is to be cleaved. Inthe present general formula, each P_(x) part of the formula refers tothe α-carbon and side chain functional group of each would be aminoacid. Thus, the P_(x) portion of an P_(x)-L_(x) pair for alaninerepresents HC—CH₃. The general formula representing the P_(x) portion ofthe general composition is —R₁CR₃—.

In general R₁ can be either CH₃ (side chain of alanine), CH(CH₃)₂ (sidechain of valine), CH₂CH(CH₃)₂ (side chain of leucine), (CH₃)CH(CH₂ CH₃)(side chain of isoleucine), CH₂(Indole) (side chain of tryptophan),CH₂(Benzene) (side chain of phenylalanine), CH₂CH₂SCH₃ (side chain ofmethionine), H (side chain of glycine), CH₂OH (side chain of serine),CHOHCH₃ (side chain of threonine), CH₂(Phenol) (side chain of tyrosine),CH₂SH (side chain of cysteine), CH₂CH₂CONH₂ (side chain of glutamine),CH₂CONH₂ (side chain of asparagine), CH₂CH₂CH₂CH₂NH₂ (side chain oflysine), CH₂CH₂CH₂NHC(NH)(NH₂) (side chain of arginine), CH₂(Imidazole)(side chain of histidine), CH₂COOH (side chain of aspartic acid),CH₂CH₂COOH (side chain of glutamic acid), and functional natural andnon-natural derivatives or synthetic substitutions of these.

It is most preferred that R₃ is a single H. In general, however, R₃ canbe alkenyl, alkynal, alkenyloxy, and alkynyloxy groups that allowbinding to memapsin 2. Preferably, alkenyl, alkynyl, alkenyloxy andalkynyloxy groups have from 2 to 40 carbons, and more preferably from 2to 20 carbons, from 2 to 10 carbons, or from 2 to 3 carbons, andfunctional natural and non-natural derivatives or syntheticsubstitutions of these.

The L_(x) portion of the P_(x)-L_(x) pair represents the atoms linkingthe P_(x) regions together. In a natural substrate the L_(x) representsthe β-carbon attached to the amino portion of what would be the nextamino acid in the chain. Thus, L_(x) would be represented by —CO—NH—.The general formula for L_(x) is represented by R₂. In general R₂ can beCO—HN (amide), CH(OH)(CH₂) (hydroxyethylene), CH(OH)CH(OH)(dihydroxyethylene), CH(OH)CH₂NH (hydroxyethylamine), PO(OH)CH₂(phosphinate), CH₂NH, (reduced amide). It is understood that more thanone L- maybe an isostere as long as the substrate analog functions toinhibit aspartic protease function.

Ls which are not isosteres may either be an amide bond or mimetic of anamide bond that is non-hydrolyzable.

X and Y represent molecules which are not typically involved in therecognition by the aspartic protease recognition site, but which do notinterfere with recognition. It is preferred that these molecules conferresistance to the degradation of the substrate analog. Preferredexamples would be amino acids coupled to the substrate analog through anon-hydrolyzable bond. Other preferred compounds would be cappingagents. Still other preferred compounds would be compounds which couldbe used in the purification of the substrate analogs such as biotin.

As used herein, alkyl refers to substituted or unsubstituted straight,branched or cyclic alkyl groups; and alkoxyl refers to substituted orunsubstituted straight, branched or cyclic alkoxy. Preferably, alkyl andalkoxy groups have from 1 to 40 carbons, and more preferably from 1 to20 carbons, from 1 to 10 carbons, or from 1 to 3 carbons.

As used herein, alkenyl refers to substituted or unsubstituted straightchain or branched alkenyl groups; alkynyl refers to substituted orunsubstituted straight chain or branched alkynyl groups; alkenyloxyrefers to substituted or unsubstituted straight chain or branchedalkenyloxy; and alkynyloxy refers to substituted or unsubstitutedstraight chain or branched alkynyloxy. Preferably, alkenyl, alkynyl,alkenyloxy and alkynyloxy groups have from 2 to 40 carbons, and morepreferably from 2 to 20 carbons, from 2 to 10 carbons, or from 2 to 3carbons.

As used herein, alkaryl refers to an alkyl group that has an arylsubstituent; aralkyl refers to an aryl group that has an alkylsubstituent; heterocyclic-alkyl refers to a heterocyclic group with analkyl substituent; alkyl-heterocyclic refers to an alkyl group that hasa heterocyclic substituent.

The substituents for alkyl, alkenyl, alkynyl, alkoxy, alkenyloxy, andalkynyloxy groups can be halogen, cyano, amino, thio, carboxy, ester,ether, thioether, carboxamide, hydroxy, or mercapto. Further, the groupscan optionally have one or more methylene groups replaced with aheteroatom, such as O, NH or S.

A number of different substrates were tested and analyzed, and thecleavage rules for Memapsin 2 were determined. The results of thesubstrates which were analyzed are presented in Table 1 and the rulesdetermined from these results are summarized below.

(1) The primary specificity site for a memapsin 2 substrate is subsiteposition, P₁′. This means that the most important determinant forsubstrate specificity in memapsin 2 is the amino acid, S1′. P₁′ mustcontain a small side chain for memapsin 2 to recognize the substrate.Preferred embodiments are substrate analogs where R₁ of the P₁′ positionis either H (side chain of glycine), CH₃ (side chain of alanine), CH₂OH(side chain of serine), or CH₂OOH (side chain of aspartic acid).Embodiments that have an R1 structurally smaller than CH₃ (side chain ofalanine) or CH₂OH (side chain of serine) are also preferred.(2) There are no specific sequence requirements at positions P₄, P₃, P₂,P₁, P₂′, P₃′, and P₄′ Each site can accommodate any other amino acidresidue in singularity as long as rule number 3 is met.(3) At least two of the remaining seven positions, P₄, P₃, P₂, P₁, P₂′,P₃′, and P₄′, must have an R₁ which is made up of a hydrophobic residue.It is preferred that there are at least three hydrophobic residues inthe remaining seven positions, P₄, P₃, P₂, P₁, P₂′, P₃′, and P₄′.Preferred R₁ groups for the positions that contain a hydrophobic groupare CH₃ (side chain of alanine), CH(CH₃)₂ (side chain of valine),CH₂CH(CH₃)₂ (side chain of leucine), (CH₃)CH(CH₂ CH₃) (side chain ofisoleucine), CH₂(INDOLE) (side chain of tryptophan), CH₂(Benzene) (sidechain of phenylalanine), CH₂CH₂SCH₃ (side chain of methionine)CH₂(Phenol) (side chain of tyrosine). It is more preferred that thehydrophobic group be a large hydrophobic group. Preferred R₁s whichcontain large hydrophobic groups are CH(CH₃)₂ (side chain of valine),CH₂CH(CH₃)₂ (side chain of leucine), (CH₃)CH(CH₂ CH₃) (side chain ofisoleucine), CH₂(Indole) (side chain of tryptophan), CH₂(Benzene) (sidechain of phenylalanine), CH₂CH₂SCH₃ (side chain of methionine)CH₂(Phenol) (side chain of tyrosine). It is most preferred thatpositions with a hydrophobic R₁ are CH(CH₃)₂ (side chain of valine),CH₂CH(CH₃)₂ (side chain of leucine), CH₂(Benzene) (side chain ofphenylalanine), CH₂CH₂SCH₃ (side chain of methionine), or CH₂(Phenol)(side chain of tyrosine).(4) None of the eight positions, P₄, P₃, P₂, P₁, P₁′, P₂′, P₃′, and P₄′may have a proline side chain at its R1 position.(5) Not all subsites must have an P represented in the analog. Forexample, a substrate analog could haveX—P₂-L₁-P₂-L₀-P₁′-L₁′-P₂′-L₂′-P₃′-L₃′-Y or it could haveX-L₁-P₁-L₀-P₁′-L₁′-P₂′-L₂′-P₃′-L₃′-P₄′L₄′-Y.

Preferred substrate analogs are analogs having the sequences disclosedin Table 1, with the non-hydrolyzable analog between P1 and P1′.

Combinatorial Chemistry to Make Inhibitors

Combinatorial chemistry includes but is not limited to all methods forisolating molecules that are capable of binding either a small moleculeor another macromolecule. Proteins, oligonucleotides, andpolysaccharides are examples of macromolecules. For example,oligonucleotide molecules with a given function, catalytic orligand-binding, can be isolated from a complex mixture of randomoligonucleotides in what has been referred to as “in vitro genetics”(Szostak, TIBS 19:89, 1992). One synthesizes a large pool of moleculesbearing random and defined sequences and subjects that complex mixture,for example, approximately 10¹⁵ individual sequences in 100 μg of a 100nucleotide RNA, to some selection and enrichment process. Throughrepeated cycles of affinity chromatography and PCR amplification of themolecules bound to the ligand on the column, Ellington and Szostak(1990) estimated that 1 in 10¹⁰ RNA molecules folded in such a way as tobind a small molecule dyes. DNA molecules with such ligand-bindingbehavior have been isolated as well (Ellington and Szostak, 1992; Bocket al, 1992).

Techniques aimed at similar goals exist for small organic molecules,proteins and peptides and other molecules known to those of skill in theart. Screening sets of molecules for a desired activity whether based onlibraries of small synthetic molecules, oligonucleotides, proteins orpeptides is broadly referred to as combinatorial chemistry.

There are a number of methods for isolating proteins either have de novoactivity or a modified activity. For example, phage display librarieshave been used for a number of years. A preferred method for isolatingproteins that have a given function is described by Roberts and Szostak(Roberts R. W. and Szostak J. W. Proc. Natl. Acad. Sci. USA,94(23)12997-302 (1997). Another preferred method for combinatorialmethods designed to isolate peptides is described in Cohen et al. (CohenB. A., et al., Proc. Natl. Acad. Sci. USA 95(24):14272-7 (1998)). Thismethod utilizes a modified two-hybrid technology. Yeast two-hybridsystems are useful for the detection and analysis of protein:proteininteractions. The two-hybrid system, initially described in the yeastSaccharomyces cerevisiae, is a powerful molecular genetic technique foridentifying new regulatory molecules, specific to the protein ofinterest (Fields and Song, Nature 340:245-6 (1989)). Cohen et al.,modified this technology so that novel interactions between synthetic orengineered peptide sequences could be identified which bind a moleculeof choice. The benefit of this type of technology is that the selectionis done in an intracellular environment. The method utilizes a libraryof peptide molecules that attach to an acidic activation domain. Apeptide of choice, for example an extracellular portion of memapsin 2 isattached to a DNA binding domain of a transcriptional activationprotein, such as Gal 4. By performing the Two-hybrid technique on thistype of system, molecules that bind the extracellular portion ofmemapsin 2 can be identified.

Screening of Small Molecule Libraries

In addition to these more specialized techniques, methodology well knownto those of skill in the art, in combination with various small moleculeor combinatorial libraries, can be used to isolate and characterizethose molecules which bind to or interact with the desired target,either memapsin 2 or its substrate. The relative binding affinity ofthese compounds can be compared and optimum inhibitors identified usingcompetitive or non-competitive binding studies which are well known tothose of skill in the art. Preferred competitive inhibitors arenon-hydrolyzable analogs of memapsin 2. Another will cause allostericrearrangements which prevent memapsin 2 from functioning or foldingcorrectly.

Computer Assisted Rational Drug Design

Another way to isolate inhibitors is through rational design. This isachieved through structural information and computer modeling. Computermodeling technology allows visualization of the three-dimensional atomicstructure of a selected molecule and the rational design of newcompounds that will interact with the molecule. The three-dimensionalconstruct typically depends on data from x-ray crystallographic analysesor NMR imaging of the selected molecule. The molecular dynamics requireforce field data. The computer graphics systems enable prediction of howa new compound will link to the target molecule and allow experimentalmanipulation of the structures of the compound and target molecule toperfect binding specificity. For examples, using NMR spectroscopy,Inouye and coworkers were able to obtain the structural information ofN-terminal truncated TSHK (transmembrane sensor histidine kinases)fragments which retain the structure of the individual sub-domains ofthe catalytic site of a TSHK. On the basis of the NMR study, they wereable to identify potential TSHK inhibitors (U.S. Pat. No. 6,077,682 toInouye). Another good example is based on the three-dimensionalstructure of a calcineurin/FKBP12/FK506 complex determined using highresolution X-ray crystallography to obtain the shape and structure ofboth the calcineurin active site binding pocket and the auxiliaryFKBP12/FK506 binding pocket (U.S. Pat. No. 5,978,740 to Armistead). Withthis information in hand, researchers can have a good understanding ofthe association of natural ligands or substrates with the bindingpockets of their corresponding receptors or enzymes and are thus able todesign and make effective inhibitors.

Prediction of molecule-compound interaction when small changes are madein one or both requires molecular mechanics software and computationallyintensive computers, usually coupled with user-friendly, menu-driveninterfaces between the molecular design program and the user. Examplesof molecular modeling systems are the CHARMm and QUANTA programs,Polygen Corporation, Waltham, Mass. CHARMm performs the energyminimization and molecular dynamics functions. QUANTA performs theconstruction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive withspecific proteins, such as Rotivinen, et al., 1988 Acta PharmaceuticaFennica 97, 159-166; Ripka, New Scientist 54-57 (Jun. 16, 1988);McKinaly and Rossmann, 1989 Annu. Rev. Pharmacol. Toxiciol. 29, 111-122;Perry and Davies, QSAR: Quantitative Structure-Activity Relationships inDrug Design pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989Proc. R. Soc. Lond. 236, 125-140 and 141-162; and, with respect to amodel enzyme for nucleic acid components, Askew, et al., 1989 J. Am.Chem. Soc. 111, 1082-1090. Other computer programs that screen andgraphically depict chemicals are available from companies such asBioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga, Ontario,Canada, and Hypercube, Inc., Cambridge, Ontario.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichalter substrate binding or enzymatic activity.

Screening of Libraries

Design of substrate analogs and rational drug design are based onknowledge of the active site and target, and utilize computer softwareprograms that create detailed structures of the enzyme and itssubstrate, as well as ways they interact, alone or in the presence ofinhibitor. These techniques are significantly enhanced with x-raycrystallographic data in hand. Inhibitors can also be obtained byscreening libraries of existing compounds for those which inhibit thecatalytically active enzyme. In contrast to reports in the literaturerelating to memapsin 2, the enzyme described herein has activityanalogous to the naturally produced enzyme, providing a means foridentifying compounds which inhibit the endogenous activity. Thesepotential inhibitors are typically identified using high throughputassays, in which enzyme, substrate (preferably a chromogemic substrate)and potential inhibitor (usually screened across a range ofconcentrations) are mixed and the extent of cleavage of substratedetermined. Potentially useful inhibitors are those which decrease theamount of cleavage.

II. Methods of Diagnosis and Treatment

Inhibitors can be used in the diagnosis and treatment and/or preventionof Alzheimer's disease and conditions associated therewith, such aselevated levels of the forty-two amino acid peptide cleavage product,and the accumulation of the peptide in amyeloid plaques.

Diagnostic Uses

The substrate analogs can be used as reagents for specifically bindingto memapsin 2 or memapsin 2 analogs and for aiding in memapsin 2isolation and purification or characterization, as described in theexamples. The inhibitors and purified recombinant enzyme can be used inscreens for those individuals more genetically prone to developAlzheimer's disease.

Therapeutic Uses

Recombinant human memapsin 2 cleaves a substrate with the sequenceLVNM/AEGD (SEQ ID NO:9). This sequence is the in vivo processing sitesequence of human presenilins. Both presenilin 1 and presenilin 2 areintegral membrane proteins. They are processed by protease cleavage,which removes the N terminal sequence from the unprocessed form. Onceprocessed, presenilin forms a two-chain heterodimer (Capell et al., J.Biol. Chem. 273, 3205 (1998); Thinakaran et al., Neurobiol. Dis. 4, 438(1998); Yu et al., Neurosci Lett. 2; 254(3):125-8 (1998)), which isstable relative to the unprocessed presenilins. Unprocessed presenilinesare quickly degraded (Thinakaran et al., J. Biol. Chem. 272, 28415(1997); Steiner et al., J. Biol. Chem. 273, 32322 (1998)). It is knownthat presenilin controls the in vivo activity of beta-secretase, whichin turn cleaves the amyloid precursor protein (APP) leading to theformation of alpha-beta42. The accumulation of alpha-beta42 in the braincells is known to be a major cause of Alzheimer's disease (for review,see Selkoe, 1998). The activity of presenilin therefore enhances theprogression of Alzheimer's disease. This is supported by the observationthat in the absence of presenilin gene, the production of alpha-beta42peptide is lowered (De Strooper et al., Nature 391, 387 (1998)). Sinceunprocessed presenilin is degraded quickly, the processed, heterodimericpresenilin must be responsible for the accumulation of alpha-beta42leading to Alzheimer's disease. The processing of presenilin by memapsin2 would enhance the production of alpha-beta42 and therefore, furtherthe progress of Alzheimer's disease. Therefore a memapsin 2 inhibitorthat crosses the blood brain barrier can be used to decrease thelikelihood of developing or slow the progression of Alzheimer's diseasewhich is mediated by deposition of alpha-beta42. Since memapsin 2cleaves APP at the beta cleavage site, prevention of APP cleavage at thebeta cleavage site will prevent the build up of alpha-beta42.

Vaccines

The catalytically active memapsin 2 or fragments thereof including theactive site defined by the presence of two catalytic aspartic residuesand substrate binding cleft can be used to induce an immune response tothe memapsin 2. The memapsin 2 is administered in an amount effective toelicit blocking antibodies, i.e., antibodies which prevent cleavage ofthe naturally occurring substrate of memapsin 2 in the brain. Anunmodified vaccine may be useful in the prevention and treatment ofAlzheimer's disease. The response to the vaccine may be influenced byits composition, such as inclusion of an adjuvant, viral proteins fromproduction of the recombinant enzyme, and/or mode of administration(amount, site of administration, frequency of administration, etc).Since it is clear that the enzyme must be properly folded in order to beactive, antibody should be elicited that is active against theendogenous memapsin 2. Antibodies that are effective against theendogenous enzyme are less likely to be produced against the enzyme thatis not property refolded.

Pharmaceutically Acceptable Carriers

The inhibitors will typically be administered orally or by injection.Oral administration is preferred. Alternatively, other formulations canbe used for delivery by pulmonary, mucosal or transdermal routes. Theinhibitor will usually be administered in combination with apharmaceutically acceptable carrier. Pharmaceutical carriers are knownto those skilled in the art. The appropriate carrier will typically beselected based on the mode of administration. Pharmaceuticalcompositions may also include one or more active ingredients such asantimicrobial agents, antiinflammatory agents, and analgesics.

Preparations for parenteral administration or administration byinjection include sterile aqueous or non-aqueous solutions, suspensions,and emulsions. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Preferred parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles include fluid andnutrient replenishers, and electrolyte replenishers (such as those basedon Ringer's dextrose).

Formulations for topical (including application to a mucosal surface,including the mouth, pulmonary, nasal, vaginal or rectal) administrationmay include ointments, lotions, creams, gels, drops, suppositories,sprays, liquids and powders. Formulations for these applications areknown. For example, a number of pulmonary formulations have beendeveloped, typically using spray drying to formulate a powder havingparticles with an aerodynanmic diameter of between one and threemicrons, consisting of drug or drug in combination with polymer and/orsurfactant.

Compositions for oral administration include powders or granules,suspensions or solutions hi water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Peptides as described herein can also be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Dosages

Dosing is dependent on severity and responsiveness of the condition tobe treated, but will normally be one or more doses per day, with courseof treatment lasting from several days to several months or until theattending physician determines no further benefit will be obtained.Persons of ordinary skill can determine optimum dosages, dosingmethodologies and repetition rates.

The dosage ranges are those large enough to produce the desired effectin which the symptoms of the memapsin 2 mediated disorder are alleviated(typically characterized by a decrease in size and/or number of amyloidplaque, or by a failure to increase in size or quantity), or in whichcleavage of the alpha-beta42 peptide is decreased. The dosage can beadjusted by the individual physician in the event of any counterindications.

The present invention will be further understood by reference to thefollowing non-limiting examples.

Example 1 Cloning of Memapsin 2 1. Cloning and Nucleotide Sequence ofPro-Memapsin 2

New sequences homologous to human aspartic proteases were found in thefollowing entries in the EST IMAGE database: AA136368 pregnant uterusATCC 947471, AA207232 neurepithelium ATCC 214526, and R55398 humanbreast ATCC 392689. The corresponding bacterial strains: #947471,#214526, and #392689 containing the EST sequences were obtained from theATCC (Rockville, Md.). The sequencing of these clones obtained from ATCCconfirmed that they contained sequences not identical to known humanaspartic proteases. The completed sequences of these clones assembledinto about 80% of prepro-M2 cDNA. Full length cDNAs of these clones wereobtained using the following methods.

The Human Pancreas Marathon-Ready cDNA (Clontech), which isdouble-strand cDNA obtained by reverse-transcription, primer addition,and second strand synthesize of mRNA from human tissues, was used astemplate for PCR amplification. An adapter primer (AP1) and a nestedadapter primer (AP2) were used for 5′ and 3′-RACE PCR. For PCR the5′-region of the memapsin 2 cDNA, primers AP1 and NHASPR1 were used.Primers for the 3′-end of the cDNA are NHASPF2 and AP1. The middle ofthe cDNA was amplified by primers NHASPF1 and NHASPR2. The sequence forthe primers is as follows: NHASPF1: GGTAAGCATCCCCCATGGCCCCAACGTC (SEQ IDNO:10),

NHASPR1: GACGTTGGGGCCATGGGGGATGCTTACC (SEQ ID NO:11),

NHASPF2: ACGTTGTCTTTGATCGGGCCCGAAAACGAATTGG (SEQ ID NO:12),

NHASPR2: CCAATTCGTTTTCGGGCCCGATCAAAGACAACG (SEQ ID NO:13),

AP1: CCATCCTAATACGACTCACTATAGGGC (SEQ ID NO:14), and

AP2: ACTCACTATAGGGCTCGAGCGGC (SEQ ID NO:15)

Memapsin 2 was also cloned from a human pancreas library (Quick-ScreenHuman cDNA Library Panel) contained in lambda-gt10 and lambda-gt11vectors. The primers from the vectors, GT10FWD, GT10REV, GT11FWD, andGT11REV, were used as outside primers. The sequence of the primers usedwas:

GT10FWD: CTTTTGAGCAAGTTCAGCCTGGTTAA, (SEQ ID NO: 16) GT10REV:GAGGTGGCTTATGAGTATTTCTTCCAGGGTA, (SEQ ID NO: 17) GT11FWD:TGGCGACGACTCCTGGAGCCCG, (SEQ ID NO: 18) GT11REV:TGACACCAGACCAACTGGTAATGG. (SEQ ID NO: 19)

In addition, memapsin 2 cDNA was amplified directly from the humanpancreatic lambda-gt10 and lambda-gt11 libraries. The sequence of theprimers was: PASPN1: catatgGCGGGAGTGCTGCCTGCCCAC (SEQ ID NO:20) and

NHASPC1: ggatccTCACTTCAGCAGGGAGATGTCATCAGCAAAGT (SEQ ID NO:21).

The amplified memapsin 2 fragments were cloned into an intermediate PCRvector (Invitrogen) and sequenced.

The assembled cDNA from the fragments, the nucleotide and the deducedprotein sequence are shown in SEQ ID NO 1 and SEQ ID NO 2.

Pro-memapsin 2 is homologous to other human aspartic proteases. Based onthe alignments, Pro-memapsin 2 contains a pro region, an asparticprotease region, and a trans-membrane region near the C-terminus. Theactive enzyme is memapsin 2 and its pro-enzyme is pro-memapsin 2.

Example 2 Distribution of Memapsin 2 in Human Tissues

Multiple tissue cDNA panels from Clontech were used as templates for PCRamplification of a 0.82 kb fragment of memapsin 2 cDNA. The primers usedfor memapsin 2 were NHASPF1 and NHASPR2. Tissues that contain memapsin 2or fragments of memapsin 2 yielded amplified PCR products. The amount ofamplified product indicated that memapsin 2 is present in the followingorgans from most abundant to least abundant: pancreas, brain, lung,kidney, liver, placenta, and heart. Memapsin 2 is also present inspleen, prostate, testis, ovary, small intestine, and colon cells.

Example 3 Expression of Pro-Memapsin 2 cDNA in E. coli, Refolding andPurification of Pro-Memapsin 2

The pro-memapsin 2 was PCR amplified and cloned into the BamHI site of apET11a vector. FIG. 1 shows the construction of two expression vectors,pET11-memapsin 2-T1 (hereafter T1) and pET11-memapsin 2-T2 (hereafterT2). In both vectors, the N-terminal 15 residues of the expressedrecombinant proteins are derived from the expression vector.Pro-memapsin 2 residues start at residue Ala-16. The two recombinantpro-memapsin 2s have different C-terminal lengths. Clone T1 ends atThr-456 and clone T2 ends at Ala-421. The T1 construct contains aC-terminal extension from the T2 construct but does not express any ofthe predicted transmembrane domain.

Expression of Recombinant Proteins and Recovery of Inclusion Bodies

The T1 and T2 expression vectors were separately transfected into E.coli strain BL21(DE3). The procedures for the culture of transfectedbacteria, induction for synthesis of recombinant proteins and therecovery and washing of inclusion bodies containing recombinant proteinsare essentially as previously described (Lin et al., 1994).

Three different refolding methods have produced satisfactory results.

(i) The Rapid Dilution Method.

Pro-memapsin 2 in 8 M urea/100 mM beta-mercaptoethanol withOD_(280 nm)=5 was rapidly diluted into 20 volumes of 20 mM-Tris, pH 90.The solution was slowly adjusted into pH 8 with 1 M HCl. The refoldingsolution was then kept at 4° C. for 24 to 48 hours before proceedingwith purification.

(ii) The Reverse Dialysis Method

An equal volume of 20 mM Tris, 0.5 mM oxidized/1.25 mM reducedglutathione, pH 9.0 is added to rapidly stirred pro-memapsin 2 in 8 Murea/10 mM beta-mercaptoethanol with OD_(280 nm)=5. The process isrepeated three more times with 1 hour intervals. The resulting solutionis then dialyzed against sufficient volume of 20 mM Tris base so thatthe final urea concentration is 0.4 M. The pH of the solution is thenslowly adjusted to 8.0 with 1 M HCl.

iii. The Preferred Method for Refolding.

Inclusion bodies are dissolved in 8 M urea, 0.1 M Tris, 1 nm Glycine, 1mM EDTA, 100 mM beta-mercaptoethanol, pH 10.0. The OD₂₈₀ of theinclusion bodies are adjusted to 5.0 with the 8 M urea solution withoutbeta-mercaptoethanol. The final solution contains the following reducingreagents: 10 mM beta-mercaptoethanol, 10 mM DTT (Dithiothreitol), 1 mMreduced glutathion, and 0.1 M oxidized glutathion. The final pH of thesolution is 10.0.

The above solution is rapidly diluted into 20 volumes of 20 mM Trisbase, the pH is adjusted to 9.0, and the resulting solution is kept at4° C. for 16 hr. The solution is equilibrated to room temperature in 6hr, and the pH is adjusted to 8.5. The solution is returned to 4° C.again for 18 hr.

The solution is again equilibrated to room temperature in 6 hr, and thepH is adjusted to 8.0. The solution is returned to 4° C. again for 4 to7 days.

The refolding procedures are critical to obtain an enzymically activepreparation which can be used for studies of subsite specificity of M2,to analyze inhibition potency of M2 inhibitors, to screen for inhibitorsusing either random structural libraries or existing collections ofcompound libraries, to produce crystals for crystallography studies ofM2 structures, and to produce monoclonal or polygonal antibodies of M2.

Purification of Recombinant Pro-Memapsin 2-T2

The refolded material is concentrated by ultrafiltration, and separatedon a SEPHACRYL™ 300 column equilibrated with 20 mM Tris.HCl, 0.4 M urea,pH 8.0. The refolded peak (second peak) from the S-300 column can befurther purified with a FPLC RESOURCE-Q™ column, which is equilibratedwith 20 mM Tris-HCl, 0.4 M urea, pH 8.0. The enzyme is eluted from thecolumn with a linear gradient of NaCl. The refolded peak from S-300 canalso be activated before further purification. For activation, thefractions are mixed with equal volume 0.2 M Sodium Acetate, 70%glycerol, pH 4.0. The mixture is incubated at 22° C. for 18 hr, and thendialyzed twice against 20 volumes of 20 mM Bis-Tris, 0.4 M urea, pH 6.0.The dialyzed materials are then further purified on a FPLC RESOURCE-Q™column equilibrated with 20 Bis-Tris, 0.4 M urea, pH 6.0. The enzyme iseluted with a linear gradient of NaCl.

SDS-PAGE analysis of the S-300 fractions under reduced and non-reducedconditions indicated that Pro-memapsin 2 first elutes as a very highmolecular weight band (greater than about 42 kD) under non-reducedconditions. This indicates that the protein is not folded properly inthese fractions, due to disulfide cross linking of proteins. Subsequentfractions contain a protein of predicted pro-memapsin 2-T2 size (about42 kDa). The pro-enzyme obtained in these fractions is alsoproteolytically active for auto-catalyzed activation. These fractionswere pooled and subjected to chromatography on the FPLC RESOURCE™ columneluted with a linear gradient of NaCl. Some fractions were analyzedusing SDS-PAGE under non-reducing conditions. The analysis showed thatfractions 6 and 7 contained most of the active proteins, which wasconsistent with the first FPLC peak containing the active protein. Themain peak was coupled to a shoulder peak, and was present with repeatedpurification with the same RESOURCE™ Q column. The main shoulder peakswere identified as active pro-memapsin 2 that exist in differentconformations under these conditions.

Example 4 Proteolytic Activity and Cleavage-Site Preferences ofRecombinant Memapsin 2

The amino acid sequence around the proteolytic cleavage sites wasdetermined in order to establish the specificity of memapsin 2.Recombinant pro-memapsin 2-T1 was incubated in 0.1 M sodium acetate, pH4.0, for 16 hours at room temperature in order to create autocatalyzedcleavages. The products were analyzed using SDS-polyacrylamide gelelectrophoresis. Several bands which corresponded to molecular weightssmaller than that of pro-memapsin 2 were observed. The electrophoreticbands were trans-blotted onto a PVDF membrane. Four bands were chosenand subjected to N-terminal sequence determination in a ProteinSequencer. The N-terminal sequence of these bands established thepositions of proteolytic cleavage sites on pro-memapsin 2.

In addition, the oxidized β-chain of bovine insulin and two differentsynthetic peptides were used as substrates for memapsin 2 to determinethe extent of other hydrolysis sites. These reactions were carried outby auto-activated pro-memapsin 2 in 0.1 M sodium acetate, pH 4.0, whichwas then incubated with the peptides. The hydrolytic products weresubjected to HPLC on a reversed phase C-18 column and the eluent peakswere subjected to electrospray mass spectrometry for the determinationof the molecular weight of the fragments. Two hydrolytic sites wereidentified on oxidized insulin B-chain (Table 1). Three hydrolytic siteswere identified from peptide NCH-gamma. A single cleavage site wasobserved in synthetic peptide PS1-gamma, whose sequence (LVNMAEGD) (SEQID NO:9) is derived from the beta-processing site of human presenilin 1(Table 1).

TABLE 1  Substrate Specificity of Memapsin 2 Site # Substrate P4 P3 P2P1 P1′ P2′ P3′ P4′ Comments 1 Pro-memapsin 2 R G S M A G V LSEQ ID NO: 36 (aa 12-18 of SEQ ID NO: 3) 2 G T Q H G I R L SEQ ID NO: 37(aa 23-30 of SEQ ID NO: 3) 3 S S N F A V G A SEQ ID NO: 38(aa 98-105 of SEQ ID NO: 3) 4 G L A Y A E I A SEQ ID NO: 39(aa 183-190 of SEQ ID NO: 3) 5 Oxidized insulin H L C{circumflex over( )} G S H L V SEQ ID NO: 22 B-chain′ 6 C{circumflex over ( )} G E R G FF Y SEQ ID NO: 23 C{circumflex over ( )} is cysteic acid 7 Synthetic  VG S G V SEQ ID NO: 24 peptide 8 V G S G V L L SEQ ID NO: 25SEQ ID NO: 26 9 G V L L S R K Three sites cleaved in a peptide:VGSGVLLSRK (SEQ ID NO: 30) 10 Peptide L V N M A E G D SEQ ID NO: 9

Example 5 Activation of Pro-Memapsin 2 and Enzyme Kinetics

Incubation in 0.1 M sodium acetate, pH 4.0, for 16 h at 22° C.auto-catalytically converted pro-M2_(pd) to M2_(pd). For initialhydrolysis tests, two synthetic peptides were separately incubated withpro-M2_(pd) in 0.1 M Na acetate, pH 4.0 for different periods rangingfrom 2 to 18 h. The incubated samples were subjected to LC/MS for theidentification of the hydrolytic products. For kinetic studies, theidentified HPLC (Beckman System Gold) product peaks were integrated forquantitation. The K_(m) and k_(cat) values for presenilin 1 and SwedishAPP peptides (Table 1) were measured by steady-state kinetics. Theindividual K_(m) and k_(cat) values for APP peptide could not bemeasured accurately by standard methods, so its k_(cat)/K_(m) value wasmeasured by competitive hydrolysis of mixed substrates againstpresenilin 1 peptide (Fersht, A. “Enzyme Structure and Mechanism”,2^(nd) Ed., W.H. Freeman and Company, New York. (1985)).

The results are shown in FIGS. 2A and 2B. The conversion of pro-M2_(pd)at pH 4.0 to smaller fragments was shown by SDS-polyacrylamideelectrophoresis. The difference in migration between pro-M2_(pd) andconverted enzyme is evident in a mixture of the two. FIG. 2A is a graphof the initial rate of hydrolysis of synthetic peptide swAPP (seeTable 1) by M2_(pd) at different pH. FIG. 2B is a graph of the relativek_(cat)/K_(m) values for steady-state kinetic of hydrolysis of peptidesubstrates by M2_(pd).

Example 6 Expression in Mammalian Cells

Methods

PM2 cDNA was cloned into the EcoRV site of vector pSecTag A(Invitrogen). Human APP cDNA was PCR amplified from human placenta8-gt11 library (Clontech) and cloned into the NheI and XbaI sites ofpSecTag A. The procedure for transfection into HeLa cells and vacciniavirus infection for T7-based expression are essentially the same asdescribed by Lin, X., FASEB J. 7:1070-1080 (1993).

Transfected cells were metabolically labeled with 200 microCi ³⁵Smethionine and cysteine (TransLabel; ICN) in 0.5 ml ofserum-free/methionine-free media for 30 min, rinsed with 1 ml media, andreplaced with 2 ml DMEM/10% FCS. In order to block vesicleacidification, Bafilomycin A1 was included in the media (Perez, R. G.,et al., J Biol. Chem 271:9100-9107 (1996)). At different time points(chase), media was removed and the cells were harvested and lysed in 50mM Tris, 0.3 M NaCl, 5 mM EDTA, 1% Triton X-100, pH 7.4, containing 10mM iodoacetamide, 10:M TPCK, 10:M TLCK, and 2 microg/ml leupeptin. Thesupernatant (14,000×g) of cell lysates and media were immunoadsorbedonto antibody bound to protein G sepharose (Sigma). Anti-APP N-terminaldomain antibody (Chemicon) was used to recover the betaN-fragment of APPand anti-alpha-beta₁₋₁₇ antibody (Chemicon, recognizing the N-terminal17 residues of alpha-beta) was used to recover the 12 kDa β C-fragment.The former antibody recognized only denatured protein, so media wasfirst incubated in 2 mM dithiothrietol 0.1% SDS at 55° C. for 30 minbefore immunoabsorption. Samples were cooled and diluted with an equalvolume of cell lysis buffer before addition of anti-APP N-terminaldomain (Chemicon). Beads were washed, eluted with loading buffer,subjected to SDS-PAGE (NOVEX™) and visualized by autoradiogram orphosphorimaging (Molecular Dynamics) on gets enhanced with Amplify(Amersham). Immunodetection of the betaN-fragment was accomplished bytransblotting onto a PVDF membrane and detecting withanti-alpha-beta₁₋₁₇ and chemiluminescent substrate (Amersham).

Results.

HeLa cells transfected with APP or M2 in 4-well chamber slides werefixed with acetone for 10 min and permeabilized in 0.2% Triton X-100 inPBS for 6 min. For localizing M2, polyclonal goat anti-pro-M2_(pd)antibodies were purified on DEAE-sepharose 6B and affinity purifiedagainst recombinant pro-M2_(pd) immobilized on Affigel (BioRad).Purified anti-pro-M2_(pd) antibodies were conjugated to Alexa568(Molecular Probes) according to the manufacturer's protocol. Fixed cellswere incubated overnight with a 1:100 dilution of antibody in PBScontaining 0.1% BSA and washed 4 times with PBS. For APP, two antibodieswere used. Antibody A β₁₋₁₇ (described above) and antibody Aβ₁₇₋₄₂,which recognizes the first 26 residues following the beta-secretasecleavage site (Chemicon). After 4 PBS washes, the cells were incubatedovernight with an anti-mouse FITC conjugate at a dilution of 1:200.Cells were mounted in Prolong anti-fade reagent (Molecular Probes) andvisualized on a Leica TCS confocal laser scanning microscope.

Example 7 Design and Synthesis of 0M99-1 and OM99-2

Based on the results of specificity studies of memapsin 2, it waspredicted that good results for positions P1 and P1′ would be Leu andAla. It was subsequently determined from the specificity data that P1′preferred small residues, such as Ala and Ser. However, the crystalstructure (determined below in Example 9) indicates that this site canaccommodate a lot of larger residues. It was demonstrated that P1′ ofmemapsin 2 is the position with the most stringent specificityrequirement where residues of small side chains, such as Ala, Ser, andAsp, are preferred. Ala was selected for P1′ mainly because itshydrophobicity over Ser and Asp is favored for the penetration of theblood-brain barrier, a requirement for the design of a memapsin 2inhibitor drug for treating Alzheimer's disease. Therefore, inhibitorswere designed to place a transition-state analogue isostere between Leuand Ala (shown a Leu*Ala, where * represents the transition-stateisostere, —CH(OH)—CH₂—) and the subsite P4, P3, P2, P2′, P3′ and P4′ arefilled with the best-secretase site sequence of the Swedish mutant fromthe beta-amyloid protein. The structures of inhibitors OM99-1 and OM99-2are shown below and in FIGS. 3A and 3B, respectively:

(SEQ ID NO: 27) OM99-1: Val-Asn-Leu*Ala-Ala-Glu-Phe (SEQ ID NO: 35)OM99-2: Glu-Val-Asn-Leu*Ala-Ala-Glu-PheThe Leu*Ala dipeptide isostere was synthesized as follows:

The Leu-Ala dipeptide isostere for the M₂-inhibitor was prepared fromL-leucine. As shown in Scheme 1, L-leucine was protected as itsBOC-derivative 2 by treatment with BOC₂O in the presence of 10% NaOH indiethyl ether for 12 h. Boc-leucine 2 was then converted to Weinrebamide 3 by treatment with isobutyl chcloroformate and N-methylpiperidinefollowed by treatment of the resulting mixed anhydride withN,O-dimethylhydroxyamine

(Nahm and Weinreb, Tetrahedron Letters 1981, 32, 3815). Reduction of 3with lithium aluminum hydride in diethyl ether provided the aldehyde 4.Reaction of the aldehyde 4 with lithium propiolate derived from thetreatment of ethyl propiolate and lithium diisopropylamide afforded theacetylenic alcohol 5 as an inseparable mixture of diastereomers (5.8:1)in 42% isolated yield (Fray, Kaye and Kleinman, J. Org. Chem. 1986, 51,4828-33). Catalytic hydrogenation of 5 over Pd/BaSO₄ followed byacid-catalyzed lactonization of the resulting gamma-hydroxy ester with acatalytic amount of acetic acid in toluene at reflux, furnished thegamma-lactone 6 and 7 in 73% yield. The isomers were separated by silicagel chromatography by using 40% ethyl acetate in hexane as the eluent.

Introduction of the methyl group at C-2 was accomplished bystereoselective alkylation of 7 with methyl iodide (Scheme 2). Thus,generation of the dianion of lactone 7 with lithium hexamethyldisilazide(2.2 equivalents) in tetrahydrofuran at −78° C. (30 min) and alkylationwith methyl iodide (1.1 equivalents) for 30 min at −78° C., followed byquenching with propionic acid (5 equivalents), provided the desiredalkylated lactone 8 (76% yield) along with a small amount (less than 5%)of the corresponding epimer (Ghosh and Fidanze, 1998 J. Org. Chem. 1998,63, 6146-54). The epimeric cis-lactone was removed by columnchromatography over silica gel using a mixture (3:1) of ethyl acetateand hexane as the solvent system. The stereochemical assignment ofalkylated lactone 8 was made based on extensive ¹H-NMR NOE experiments.Aqueous lithium hydroxide promoted hydrolysis of the lactone 8 followedby protection of the gamma-hydroxyl group with tert-butyldimethylsilylchloride in the presence of imidazole and dimethylaminopyridine indimethylformamide afforded the acid 9 in 90% yield after standardwork-up and chromatography. Selective removal of the BOC-group waseffected by treatment with trifluoroacetic acid in dichloromethane at 0°C. for 1 h. The resulting amine salt was then reacted with commercial(Aldrich, Milwaukee) Fmoc-succinimide derivative in dioxane in thepresence of aqueous NaHCO₃ to provide the Fmoc-protected L*A isostere 10in 65% yield after chromatography. Protected isostere 10 was utilized inthe preparation of a random sequence inhibitor library.

Experimental Procedure

N-(tert-Butoxycarbonyl)-L-Leucine (2)

To the suspension of 10 g (76.2 mmol) of L-leucine in 140 mL of diethylether was added 80 mL of 10% NaOH. After all solid dissolves, 20 mL(87.1 mmol) of BOC₂O was added to the reaction mixture. The resultingreaction mixture was stirred at 23° C. for 12 h. After this period, thelayers were separated and the aqueous layer was acidified to pH 1 bycareful addition of 1 N aqueous HCl at 0° C. The resulting mixture wasextracted with ethyl acetate (3×100 mL). The organic layers werecombined and washed with brine and dried over anhydrous Na₂SO₄. Thesolvent was removed under reduced pressure to provide title productwhich was used directly for next reaction without further purification(yield, 97%). ¹H NMR (400 MHz, CDCl₃) δ 4.89 (broad d, 1H, J=8.3 Hz),4.31 (m, 1H), 1.74-1.49 (m, 3H), 1.44 (s, 9H), 0.95 (d, 6H, J=6.5 Hz).

N-(tert-Butoxycarbonyl)-L-leucine-N′-methoxy-N′-methyla-mide (3)

To a stirred solution of N,O-dimethylhydroxyamine hydrochloride (5.52 g,56.6 mmol) in dry dichloromethane (25 mL) under N₂ atmosphere at 0° C.,-methylpiperidine (6.9 mL, 56.6 mmol) was added dropwise. The resultingmixture was stirred at 0° C. for 30 min. In a separate flask,N-(tert-butyloxycarbonyl)-L-leucine (1) (11.9 g, 51.4 mmol) wasdissolved in a mixture of THF (45 mL) and dichloromethane (180 mL) underN₂ atmosphere. The resulting solution was cooled to −20° C. To thissolution was added 1-methylpiperidine (6.9 mL, 56.6 mmol) followed byisobutyl chloroformate (7.3 mL, 56.6 mmol). The resulting mixture wasstirred for 5 minutes at −20° C. and the above solution ofN,O-dimethylhydroxyamine was added to it. The reaction mixture was kept−20° C. for 30 minutes and then warmed to 23° C. The reaction wasquenched with water and the layers were separated. The aqueous layer wasextracted with dichloromethane (3×100 mL). The combined organic layerswere washed with 10% citric acid, saturated sodium bicarbonate, andbrine. The organic layer was dried over anhydrous Na₂SO₄ andconcentrated under the reduced pressure. The residue was purified byflash silica gel chromatography (25% ethyl acetate/hexane) to yield thetitle compound 3 (13.8 g, 97%) as a pale yellow oil. ¹H NMR (400 MHz,CDCl₃) δ 5.06 (broad d, 1H, J=9.1 Hz), 4.70 (m, 1H), 3.82 (s, 3H), 3.13(s, 3H), 1.70 (m, 1H), 1.46-1.36 (m, 2H) 1.41 (s, 9H), 0.93 (dd, 6H,J=6.5, 14.2 Hz).

N-(tert-Butoxycarbonyl)-L-leucinal (4)

To a stirred suspension of lithium aluminum hydride (770 mg, 20.3 mmol)in dry diethyl ether (60 mL) at −40° C. under N₂ atmosphere, was addedN-tert-butyloxycarbonyl-L-leucine-N′-methoxy-N′-methylamide (5.05 g,18.4 mmol) in diethyl ether (20 mL). The resulting reaction mixture wasstirred for 30 min. After this period, the reaction was quenched with10% NaHSO₄ solution (30 mL). The resulting reaction mixture was thenwarmed to 23° C. and stirred at that temperature for 30 min. Theresulting solution was filtered and the filter cake was washed by twoportions of diethyl ether. The combined organic layers were washed withsaturated sodium bicarbonate, brine and dried over anydrous MgSO₄.Evaporation of the solvent under reduced pressure afforded the titlealdehyde 4 (3.41 g) as a pale yellow oil. The resulting aldehyde wasused immediately without further purification. ¹H NMR (400 MHz, CDCl₃) δ9.5 (s, 1H), 4.9 (s, 1H), 4.2 (broad m, 1H), 1.8-1.6 (m, 2H), 1.44 (s,9H), 1.49-1.39 (m, 1H), 0.96 (dd, 6H, J=2.7, 6.5 Hz).

Ethyl (4S,5S)- and(4R,5S)-5-[(tert-Butoxycarbonyl)amino]-4-hydroxy-7-methyloct-2-ynoate(5)

To a stirred solution of diisopropylamine (1.1 mL, 7.9 mmol) in dry THF(60 mL) at 0° C. under N₂ atmosphere, was added n-BuLi (1.6 M in hexane,4.95 mL, 7.9 mmol) dropwise. The resulting solution was stirred at 0° C.for 5 min and then warmed to 23° C. and stirred for 15 min. The mixturewas cooled to −78° C. and ethyl propiolate (801 μL) in THF (2 mL) wasadded dropwise over a period of 5 min. The mixture was stirred for 30min, after which N-Boc-L-leucinal 4 (1.55 g, 7.2 mmol) in 8 mL of dryTHF was added. The resulting mixture was stirred at −78° C. for 1 h.After this period, the reaction was quenched with acetic acid (5 mL) inTHF (20 mL). The reaction mixture was warmed up to 23° C. and brinesolution was added. The layers were separated and the organic layer waswashed with saturated sodium bicarbonate and dried over Na₂SO₄.Evaporation of the solvent under reduced pressure provided a residuewhich was purified by flash silica gel chromatography (15% ethylacetate/hexane) to afford a mixture (3:1) of acetylenic alcohols 5 (0.96g, 42%). ¹H NMR (300 MHz, CDCl₃) δ 4.64 (d, 1H, J=9.0 Hz), 4.44 (broads, 1H), 4.18 (m, 2H), 3.76 (m, 1H), 1.63 (m, 1H), 1.43-1.31 (m, 2H),1.39 (s, 9H), 1.29-1.18 (m, 3H), 0.89 (m, 6H).

(5S,1′S)-5-[1′-[(tert-Butoxycarbonyl)amino]-3′-methylbutyl]-dihydrofuran-2(3H)-one(7)

To a stirred solution of the above-mixture of acetylenic alcohols (1.73g, 5.5 mmol) in ethyl acetate (20 mL) was added 5% Pd/BaSO₄ (1 g). Theresulting mixture was hydrogenated at 50 psi for 1.5 h. After thisperiod, the catalyst was filtered off through a plug of Celite and thefiltrate was concentrated under reduced pressure. The residue wasdissolved in toluene (20 mL) and acetic acid (100 μL). The reactionmixture was refluxed for 6 h. After this period, the reaction was cooledto 23° C. and the solvent was evaporated to give a residue which waspurified by flash silica gel chromatography (40% diethyl ether/hexane)to yield the (5S,1S′)-gamma-lactone 7 (0.94 g, 62.8 and the(5R,1S′)-gamma-lactone 6 (0.16 g, 10.7%). Lactone 7: ¹H NMR (400 MHz,CDCl₃) δ 4.50-4.44 (m, 2H), 3.84-3.82 (m, 1H), 2.50 (t, 2H, J=7.8 Hz),2.22-2.10 (m, 2H), 1.64-1.31 (m, 3H), 1.41 (s, 9H), 0.91 (dd, 6H, J=2.2,6.7 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 177.2, 156.0, 82.5, 79.8, 51.0, 42.2,28.6, 28.2, 24.7, 24.2, 23.0, 21.9.

(3R,5,1′S)-5-[1′-[(tert-Butoxycarbonyl)amino)]-3′-methylbut-yl]-3-methyldihydrofuran-2(3H)-one (8)

To a stirred solution of the lactone 7 (451.8 mg, 1.67 mmol) in dry THF(8 mL) at −78° C. under N₂ atmosphere, was added lithiumhexamethyldisilazane (3.67 mL, 1.0 M in THF) over a period of 3 min. Theresulting mixture was stirred at −78° C. for 30 min to generate thelithium enolate. After this period, MeI (228 μL) was added dropwise andthe resulting mixture was stirred at −78° C. for 20 min. The reactionwas quenched with saturated aqueous NH₄Cl solution and was allowed towarm to 23° C. The reaction mixture was concentrated under reducedpressure and the residue was extracted with ethyl acetate (3×100 mL).The combined organic layers were washed with brine and dried offeranhydrous Na₂SO₄. Evaporation of the solvent afforded a residue whichwas purified by silica gel chromatography (15% ethyl acetate/hexane) tofurnish the alkylated lactone 8 (0.36 g, 76%) as an amorphous solid. ¹HNMR (300 MHz, CDCl₃) δ 4.43 (broad t, 1H, J=6.3 Hz), 4.33 (d, 1H, J=9.6Hz), 3.78 (m, 1H), 2.62 (m, 1H), 2.35 (m, 1H), 1.86 (m, 1H), 1.63-1.24(m, 3H), 1.37 (s, 9H), 1.21 (d, 3H, J=7.5 Hz), 0.87 (dd, 6H, J=2.6, 6.7Hz); ¹³C NMR (75 MHz, CDCl₃) δ 180.4, 156.0, 80.3, 79.8, 51.6, 41.9,34.3, 32.5, 28.3, 24.7, 23.0, 21.8, 16.6.

(2R,4S,5S)-5-[(tert-Butoxycarbonyl)(amino]-4-[(tert-butyldimethylsilyl)oxy]-2,7-dimethyloctanoicacid (9)

To a stirred solution of lactone 8 (0.33 g, 117 mmol) in THF (2 mL) wasadded 1 N aqueous LiOH solution (5.8 mL). The resulting mixture wasstirred at 23° C. for 10 h. After this period, the reaction mixture wasconcentrated under reduced pressure and the remaining aqueous residuewas cooled to 0° C. and acidified with 25% citric acid solution to pH 4.The resulting acidic solution was extracted with ethyl acetate (3×50mL). The combined organic layers were washed with brine, dried overNa₂SO₄ and concentrated to yield the corresponding hydroxy acid (330 mg)as a white foam. This hydroxy acid was used directly for the nextreaction without further purification.

To the above hydroxy acid (330 mg, 1.1 mmol) in anhydrous DMF was addedimidazole (1.59 g, 23.34 mmol) and tert-butyldimethylchlorosilane (1.76g, 11.67 mmol). The resulting mixture was stirred at 23° C. for 24 h.After this period, MeOH (4 mL) was added and the mixture was stirred for1 h. The mixture was diluted with 25% citric acid (20 mL) and wasextracted with ethyl acetate (3×20 mL). The combined extracts werewashed with water, brine and dried over anhydrous Na₂SO₄. Evaporation ofthe solvent gave a viscous oil which was purified by flashchromatography over silica gel (35% ethyl acetate/hexane) to afford thesilyl protected acid 9 (0.44 g, 90%). IR (neat) 3300-3000 (broad), 2955,2932, 2859, 1711 cm⁻¹; ¹H NMR (400 MHz, DMSO-d⁶, 343 K) delta 6.20(broad s, 1H), 3.68 (m, 1H), 3.51 (broad s, 1H), 2.49-2.42 (m, 1H), 1.83(t, 1H, J=10.1 Hz), 1.56 (m, 1H), 1.37 (s, 9H), 1.28-1.12 (m, 3H), 1.08(d, 3H, J=7.1 Hz), 0.87 (d, 3H, J=6.1 Hz) 0.86 (s, 9H), 0.82 (d, 3H,J=6.5 Hz), 0.084 (s, 3H), 0.052 (s, 3H).

(2R,4S,5s)-5-[(fluorenylmethyloxycarbonyl)amino]-4-[(tert-butyldi-methylsilyl)oxy)]-2,7-dimethyloctanoicacid (10)

To a stirred solution of the acid 9 (0.17 g, 0.41 mmol) indichloromethane (2 mL) at 0° C. was added trifluoroacetic acid (500 mL).The resulting mixture was stirred at 0° C. for 1 h and an additionalportion (500 μL) of trifluoroacetic acid was added to the reactionmixture. The mixture was stirred for an additional 30 min and theprogress of the reaction was monitored by TLC. After this period, thesolvents were carefully removed under reduced pressure at a bathtemperature not exceeding 5° C. The residue was dissolved in dioxane (3mL) and NaHCO₃ (300 mg) in 5 mL of H₂O. To this solution was addedFmoc-succinimide (166.5 mg, 0.49 mmol) in 5 mL of dioxane. The resultingmixture was stirred at 23° C. for 8 h. The mixture was then diluted withH₂O (5 mL) and acidified with 25% aqueous citric acid to pH 4. Theacidic solution was extracted with ethyl acetate (3×50 mL). The combinedextracts were washed with brine, dried over Na₂SO₄ and concentratedunder reduced pressure to give a viscous oil residue. Purification ofthe residue by flash chromatography over silica gel afforded theFmoc-protected acid 10 (137 mg, 61%) as a white foam. ¹H NMR (400 MHz,DMSO-d⁶, 343 K) δ 7.84 (d, 2H, J=7.4 Hz), 7.66 (d, 2H, J=8 Hz), 7.39 (t,2H, J=7.4 Hz), 7.29 (m, 2H), 6.8 (s, 1H), 4.29-4.19 (m, 3H), 3.74-3.59(m, 2H), 2.49 (m, 1H), 1.88 (m, 1H), 1.58 (m, 1H), 1.31-1.17 (m, 3H),1.10 (d, 3H, J=7.1 Hz), 0.88 (s, 9H), 0.82 (d, 6H, J=6.2 Hz), 0.089 (s,3H), 0.057 (s, 3H).

The synthesis of OM99-1 and OM99-2 were accomplished using solid statepeptide synthesis procedure in which Leu*Ala was incorporated in thefourth step. The synthesized inhibitors were purified by reverse phaseHPLC and their structure confirmed by mass spectrometry.

Example 8 Inhibition of Memapsin 2 by OM99-1 and OM99-2

Enzyme activity was measured as described above, but with the additionof either OM99-1 or OM99-2. OM99-1 inhibited recombinant memapsin 2 asshown in FIG. 4A. The Ki calculated is 3×10⁻⁸ M. The substrate used wasa synthetic fluorogenic peptide substrate. The inhibition of OM99-2 onrecombinant memapsin 2 was measured using the same fluorogenicsubstrate. The Ki value was determined to be 9.58×10⁻⁹ M, as shown inFIG. 4B.

These results demonstrate that the predicted subsite specificity isaccurate and that inhibitors can be designed based on the predictedspecificity.

The residues in P1 and P1′ are very important since the M2 inhibitormust penetrate the blood-brain barrier (BBB). The choice of Ala in P1′facilitates the penetration of BBB. Analogues of Ala side chains willalso work. For example, in addition to the methyl side chain of Ala,substituted methyl groups and groups about the same size like methyl orethyl groups can be substituted for the Ala side chain. Leu at P1 canalso be substituted by groups of similar sizes or with substitutions onLeu side chain. For penetrating the BBB, it is desirable to make theinhibitors smaller. One can therefore use OM99-1 as a starting point anddiscard the outside subsites P4, P3, P3′ and P4′. The retained structureAsn-Leu*Ala-Ala (SEQ ID NO:29) is then further evolved withsubstitutions for a tight-binding M2 inhibitor which can also penetratethe BBB.

Example 9 Crystallization and X-Ray Diffraction Study of the ProteaseDomain of Human Memapsin 2 Complexed to a Specifically DesignedInhibitor, OM99-2

The crystallization condition and preliminary x-ray diffraction data onrecombinant human memapsin 2 complexed to OM99-2 were determined.

Production of Recombinant Memapsin 2

About 50 mg of recombinant memapsin 2 was purified as described inExample 3. For optimal crystal growth, memapsin 2 must be highlypurified. Memapsin 2 was over-expressed from vector pET11a-M2pd. Thismemapsin 2 is the zymogen domain which includes the pro and catalyticdomains to the end of the C-terminal extension but does not include thetransmembrane and the intracellular domains. The vector was transfectedinto E. coli BL21 (DE3) and plated onto ZB agar containing 50 mg/literampicillin. A single colony was picked to inoculate 100 ml of liquid ZBcontaining 5 mg ampicillin and cultured at 30° C., for 18 hours, withshaking at 220 RPM. Aliquots of approximately 15 ml of the overnightculture were used to inoculate each 1 liter of LB containing 50 mg ofampicillin. Cultures were grown at 37° C., with shaking at 180 RPM,until an optical density at 600 nm near 0.8 was attained. At that time,expression was induced by addition of 119 mg of IPTG to each liter ofculture. Incubation was continued for 3 additional hours post-induction.

Bacteria were harvested, suspended in 50 mM Tris, 150 μM NaCl, pH 7.5(TN buffer), and lysed by incubation with 6 mg lysozyme for 30 minutes,followed by freezing for 18 hours at −20° C. Lysate was thawed and madeto 1 mM MgCl₂ then 1000 Kunitz units of DNAse were added with stirring,and incubated for 30 min. Volume was expanded to 500 ml with TNcontaining 0.1% Triton X-100 (TNT buffer) and lysate stirred for 30minutes. Insoluble inclusion bodies containing greater than 90% memapsin2 protein were pelleted by centrifugation, and washed by resuspension inTNT with stirring for 1-2 hours. Following three additional TNT washes,the memapsin 2 inclusion bodies were dissolved in 40 ml of 8 M urea, 1mM EDTA, 1 mM glycine, 100 mM Tris base, 100 mM beta-mercaptoethanlol (8M urea buffer). Optical density at 280 nm was measured, and volumeexpanded with 8 M urea buffer to achieve final O.D. near 0.5, withaddition of sufficient quantity of beta-mercaptoethanol to attain 10 mMtotal, and 10 mM DTT, 1 mM reduced glutathione, 0.1 mM oxidizedglutathione. The pH of the solution was adjusted to 10.0 or greater, anddivided into four aliquots of 200 ml each. Each 200 ml wasrapidly-diluted into 4 liters of 20 mM Tris base, with rapid stirring.The pH was adjusted immediately to 9.0, with 1 M HCl, and stored at 4°C. overnight. The following morning the diluted memapsin 2 solution wasmaintained at room temperature for 4-6 hours followed by adjusting pH to8.5 and replacing the flasks to the 4° C. room. The same procedure wasfollowed the next day with adjustment of pH to 8.0.

This memapsin 2 solution was allowed to stand at 4° C. for 2-3 weeks.The total volume of approximately 16 liters was concentrated to 40 mlsusing ultra-filtration (Millipore) and stir-cells (Amicon), andcentrifuged at 140,000×g at 30 minutes in a rotor pre-equilbrated to 4°C. The recovered supernatant was applied to a 2.5×100 cm column of S-300equilibrated in 0.4 M urea, 20 mM Tris-HCl, pH 8.0, and eluted with thesame buffer at 30 ml/hour. The active fraction of memapsin 2 was pooledand further purified in a FPLC using a 1 ml Resource-Q (Pharmacia)column. Sample was filtered, and applied to the Resource-Q columnequilibrated in 0.4 M urea, 50 mM Tris-HCl, pH 8.0. Sample was elutedwith a gradient of 0-1 M NaCl in the same buffer, over 30 ml at 2ml/min. The eluents containing memapsin 2 appeared near 0.4 M NaCl whichwas pooled for crystallization procedure at a concentration near 5mg/ml.

The amino-terminal sequence of the protein before crystallization showedtwo sequences starting respectively at residues 28p and 30p. Apparently,the pro peptide of recombinant pro-memapsin 2 had been cleaved duringthe preparation by a yet unidentified proteolytic activity.

The activation of the folded pro-enzyme to mature enzyme, memapsin 2,was carried out as described above, i.e., incubation in 0.1 M sodiumacetate pH 4.0 for 16 hours at 22° C. Activated enzyme was furtherpurified using anion-exchange column chromatography on Resource-Q anionexchange column. The purity of the enzyme was demonstrated by SDS-gelelectrophoresis. At each step of the purification, the specific activityof the enzyme was assayed as described above to ensure the activity ofthe enzyme.

Preliminary Crystallization with OM99-2

Crystal trials were performed on purified memapsin 2 in complex with asubstrate based transition-state inhibitor OM99-2 with a Ki=10 nM.OM99-2 is equivalent to eight amino-acid residues (including subsitesS4, S3, S2, S1, S1′, S2′, S3′, and S4′ in a sequence EVNLAAEF (SEQ IDNO:28) with the substitution of the peptide bond between the S1 and S1′(L-A) by a transition-state isostere hydroxyethylene. Purified M2 wasconcentrated and mixed with a 10 fold excessive molar amount ofinhibitor. The mixture was incubated at room temperature for 2-3 hoursto optimize the inhibitor binding. The crystallization trial wasconducted at 20° C. using the hanging drop vapor diffusion procedure. Asystemic search with various crystallization conditions was conducted tofind the optimum crystallization condition for memapsin 2/OM99-2inhibitor complex. For the first step, a coarse screen aimed at coveringa wide range of potential conditions were carried out using the SparseMatrix Crystallization Screen Kits purchased from Hampton Research.Protein concentration and temperature were used as additional variables.Conditions giving promising (micro) crystals were subsequently used asstarting points for optimization, using fine grids of pH, precipitantsconcentration etc.

Crystals of memapsin-inhibitor complex were obtained at 30% PEG 8000,0.1 M NaCocadylate, pH 6.4. SDS gel electrophoresis of a dissolvedcrystal verified that the content of the crystal to be memapsin 2.Several single crystals (with the sizes about 0.3 mm×0.2 mm×0.1 mm) werecarefully removed from the cluster for data collection on a Raxis IVimage plate. These results showed that the crystals diffract to 2.6 Å.An X-ray image visualization and integration software-Denzo, was used tovisualize and index the diffraction data. Denzo identified that theprimitive orthorhombic lattice has the highest symmetry with asignificantly low distortion index. The unit cell parameters weredetermined as: a=89.1 Å, b=96.6 Å, c=134.1 Å, α=β=γ=90°. There are twomemapsin 2/OM99-2 complexes per crystallographic asymmetric unit, theV_(m) of the crystal is 2.9 Å³/Da. Diffraction extinctions suggestedthat the space group is P2₁2₁2₁.

With diffraction of the current crystal to 2.6 Å, the crystal structureobtained from these data has the potential to reach atomic solution,i.e., the three-dimensional positions of atoms and chemical bonds in theinhibitor and in memapsin 2 can be deduced. Since memapsin 2 sequence ishomologous with other mammalian aspartic proteases, e.g., pepsin orcathepsin D, it is predicted that the three dimensional structures ofmemapsin 2 will be similar (but not identical) to their structures.Therefore, in the determination of x-ray structure from the diffractiondata obtained from the current crystal, it is likely the solution of thephase can be obtained from the molecular replacement method using theknown crystal structure of aspartic proteases as the search model.

Further Crystallization Studies

Concentrated memapsin 2 was mixed with 10-fold molar excessive of theinhibitor. The mixture was incubated at room temperature for 2-3 hoursto optimize inhibitor binding, and then clarified with a 0.2 micronfilter using centrifugation. Crystals of memapsin 2-inhibitor complexwere grown at 20° C. by hanging drop vapor diffusion method using equalvolumes of enzyme-inhibitor and well solution. Crystals of qualitysuitable for diffraction studies were obtained in two weeks in 0.1 Msodium cacodylate, pH 7.4, 0.2 M (NH₄)₂SO₄, and 22.5% PEG8000. Thetypical size of the crystals was about 0.4×0.4×0.2 mm³.

Diffraction data were measured on a Raxis-IV image plate with a RigakuX-ray generator, processed with the HKL program package [Z. Otwinowski,W. Minor, Methods Enzymol. 276, 307 (1997)] A single crystal ofapproximately 0.4×0.4×0.2 mm³ in size was treated with a cryo-protectionsolution of 25% PEG8000, 20% glycerol, 0.1 M sodium-cacodylate pH 6.6,and 0.2 M (NH₄)₂SO₄, and then flash-cooled with liquid nitrogen to about−180° C. for data collection. Diffraction was observed to at least 1.9Å. The crystal form belongs to space group P2₁ with two memapsin2/OM99-2 complexes per crystallographic asymmetric unit and 56% solventcontent.

Molecular replacement was performed with data in the range of 15.0-3.5 Åusing program AmoRe, CCP4 package[Navaza, J., Acta Crystallog. Sect. A.50, 157 (1994)]. Pepsin, a human aspartic protease with 22% sequenceidentity (SEQ ID NO:31), was used as the search model(PDB id 1 psn).Rotation and transition search, followed by rigid body refinement,identified a top solution and positioned both molecules in theasymmetric unit. The initial solution had a correlation coefficient of22% and an R-factor of 0.51. The refinement was carried out using theprogram CNS [Brunger et al., Acta Crystallogr. Sect. D, 54, 905 (1998)]10% of reflections were randomly selected prior to refinement forR_(free) monitoring [Bruger, A. T., X-PLOR Version 3.1: A system forX-ray Crystallography and NMR, Yale University Press, New Haven, Conn.(1992)]. Molecular graphics program [Jones, T. A., et al., Improvedmethods for building protein models in electron density maps andlocation of errors in these models. Acta Crysallogr. Sect. A 47, 110(1991)] was used for map display and model building. From the initialpepsin model, corresponding amino acid residues were changed to that ofmemapsin 2 according to sequence alignment. The side chain conformationswere decided by the initial electron density map and a rotomer library.This model was refined using molecular dynamics and energy minimizationfunction of CNS [Bruger, A. T., et al., Acta Crystallogr. Sect. D, 54,905 (1998)]. The first cycle of refinement dropped the R_(working) to41% and the R_(free) to 45%. At this state, electron densities in theomit map clearly showed the inhibitor configuration in the active sitecleft. Structural features unique to memapsin 2 in chain tracing,secondary structure, insertions, deletions and extensions (as comparedto the search model) are identified and constructed in subsequentiterations of crystallographic refinement and map fitting. The inhibitorwas built into the corresponding electron density.

About 440 solvent molecules were then gradually added to the structureas identified in the |Fo|-|Fc| map contoured at the 3 sigma level.Non-crystallographic symmetry restriction and averaging were used inearly stages of refinement and model building. Bulk solvent andanisotropic over-all B factor corrections were applied through therefinement. The final structure was validated by the program PROCHECK,Laskowski, R. A. et al., J. Appl. Crystallog. 26, 283 (1993) whichshowed that 95% of the residues are located in the most favored regionof the Ramachandran plot. All the main chain and side chain parametersare within or better than the standard criteria. The final R_(working)and R_(free) are 18% and 22% respectively. Refinement statistics of thecrystallized memapsin 2 protein, residues 1-488 of SEQ ID NO: 2, arelisted in Table 2.

TABLE 2 Data Collection and Refinement Statistics A. A. Data StatisticsSpace group P2₁ Unit cell (a, b, and c in Å) 53.7, 85.9, 109.2 (α, β,and γ in degrees) 90.0, 101.4, 90.0 Resolution (Å) 25.0-1.9 Number ofobserved reflections 144,164 Number of unique reflections 69,056R_(merge) ^(a) 0.061 (0.25) Data completeness (%) (25.0-1.9 Å)  90.0(68.5) <I/σ(I)> 13.7 (3.0) II. B. Refinement Statistics R_(working) ^(b)0.186 R_(free) ^(b) 0.228 RMS deviation from ideal values Bond length(Å) 0.014 Bond angle (Deg) 1.7 Number of water molecules 445 AverageB-factor (Å²) Protein 28.5 Solvent 32.2 ^(a)R_(merge) =Σ_(hkl)Σ_(i)|I_(hkl,i) − <I_(hkl)>|/Σ_(hkl) <I_(hkl)>, where I_(hkl,i)is the intensity of the ith measurement and <I_(hkl)> is the weightedmean of all measurements of I_(hkl.) ^(b)R_(working (free)) = Σ||F_(o)|− |F_(c)||/Σ|F_(o)|, where F_(o) and F_(c) are the observed andcalculated structure factors. Numbers in parentheses are thecorresponding numbers for the highest resolution shell (2.00-1.9 Å).Reflections with F_(o)/σ(F_(o)) >= 0.0 are included in the refinementand R factor calculation.

Memapsin 2 Crystal Structure.

The bilobal structure of memapsin 2 (FIG. 6) is characteristic ofaspartic proteases (Tang, J., et al., Nature 271, 618-621 (1978)) withthe conserved folding of the globular core. The substrate binding cleft,where the inhibitor is bound (FIG. 6), is located between the two lobes.A pseudo two-fold symmetry between the N-(residues 1-180) (SEQ ID NO:46)and C- (residues 181-385) (SEQ ID NO:47) lobes (FIG. 6), which share 61superimposable atoms with an overall 2.3 Å rms deviation using a 4 Åcutoff. The corresponding numbers for pepsin are 67 atoms and 2.2 Å.Active-site AsP³² and Asp²²⁸ and the surrounding hydrogen-bond networkare located in the center of the cleft (FIG. 6) and are conserved withthe typical active-site conformation (Davies, D. R., Annu. Rev. Biophys.Chem. 19, 189 (1990)). The active site carboxyls are, however, notco-planar and the degree of which) (50° exceeds those observedpreviously.

Compared to pepsin, the conformation of the N-lobe is essentiallyconserved (Sielecki et al., 1990). The most significant structuraldifferences are the insertions and a C-terminal extension in the C-lobe.Four insertions in helices add loops (FIG. 6) are located on theadjacent molecular surface. Insertion F, which contains four acidicresidues, is the most negatively charged surface on the molecule.Together, these insertions enlarged significantly the molecular boundaryof memapsin 2 as compared to pepsin (FIG. 7). These surface structuralchanges may have function in the association of memapsin 2 with othercell surface components. Insertions B and E are located on the otherside of the molecule (FIG. 6). The latter contains a beta-strand thatpaired with part of the C-terminal extension G. A six- residue deletionoccurs at position 329 on a loop facing the flap on the opposite side ofthe active-site cleft, resulting in an apparently more accessible cleft.Most of the C-terminal extension (residues 359-393) (SEQ ID NO:48) is inhighly ordered structure. Residues 369-376 (SEQ ID NO:49) form a betastructure with 7 hydrogen bonds to strand 293-299 (SEQ ID NO:50), whileresidues 378-383 (SEQ ID NO:51) form a helix (FIGS. 6 and 7). Twodisulfide pairs (residues 155/359 and 217/382) unique to memapsin 2fasten both ends of the extension region to the C-lobe. This C-terminalextension is much longer than those observed previously and isconformationally different [Cutfield, S. M., et al., Structure 3, 1261(1995); Abad-Zapatero, C., et al., Protein Sci. 5, 640 (1996); Symersky,J. et al., Biochemistry 36, 12700 (1997); Yang, J., et al., ActaCrystallogr. D 55, 625 (1999)]. The last eight residues (386-393) (SEQID NO:52) are not seen in the electron density map; they may form aconnecting stern between the globular catalytic domain and the membraneanchoring domain.

Of the 21 putative pro residues only the last six, 43p-48p (SEQ IDNO:53), are visible in the electron density map. The remainders arelikely mobile. Pro-memapsin expressed in mammalian cell culture has anN-terminus position at Glu^(33p). However, an Arg-Arg sequence presentat residues 43p-44p is a frequent signal for pro-protein processing,e.g., in prorenin (Corvol, P. et al., Hypertension 5, 13-9 (1983)).Recombinant memapsin 2 derived from this cleavage is fully active. Themobility of residues 28p-42p (SEQ ID NO:54) suggests that they are notpart of the structure of mature memapsin 2.

Memapsin 2-OM99-2 Interaction.

The binding of the eight-residue inhibitor OM99-2 in the active-sitecleft shares some structural features with other asparticprotease-inhibitor complexes [Davies, D. R., Annu. Rev. Biophys. Chem.19, 189 (1990); Bailey and Cooper, (1994); Dealwis et al., (1994)].These include four hydrogen bonds between the two active-site asparticsto the hydroxyl of the transition-state isostere, the covering of theflap (residues 69-75) (SEQ ID NO:55) over the central part of theinhibitor and ten hydrogen bonds to inhibitor backbone (FIGS. 8 and 9).Most of the latter are highly conserved among aspartic proteases[Davies, D. R. Annu. Rev. Biophys. Chem. 19, 189 (1990); Bailey andCooper, (1994); Dealwis et al., (1994)] except that hydrogen bonds toGly¹¹ and Tyr¹⁹⁸ are unique to memapsin 2. These observations illustratethat the manner by which memapsin 2 transition-state template forsubstrate peptide backbone and mechanism of catalysis are similar toother aspartic proteases. These common features are, however, not thedecisive factors in the design of specific memapsin 2 inhibitors withhigh selectivity.

The observation important for the design of inhibitor drugs is that thememapsin 2 residues in contact with individual inhibitor side chains(FIG. 8) are quite different from those for other aspartic proteases.These side chain contacts are important for the design of tight bindinginhibitor with high selectivity. Five N-terminal residues of OM99-2 arein extended conformation and, with the exception of P₁′ Ala, all haveclearly defined contacts (within 4 Å of an inhibitor side chain) withenzyme residues in the active-site cleft (FIG. 8).

The protease S4 subsite is mostly hydrophilic and open to solvent. Theposition of inhibitor P₄ Glu side chain is defined by hydrogen bonds toGly¹¹ and to P₂ Asn (FIG. 8) and the nearby sidechains of Arg²³⁵ andArg³⁰⁷, which explains why the absence of this residue from OM99-2 causea 10-fold increase in K_(i). Likewise, the protease S₂ subsite isrelatively hydrophilic and open to solvent. Inhibitor P₂ Asn side chainhas hydrogen bonds to P₄ Glu and Arg²³⁵. The relatively small S₂residues Ser³²⁵ and Ser³²⁷ (Gln and Met respectively in pepsin) may fita side chain larger than Asn. Memapsin 2 S₁ and S₃ subsites, whichconsist mostly of hydrophobic residues, have conformations verydifferent from pepsin due to the deletion of pepsin helix h_(H2)(Dealwis, et al., (1994)). The inhibitor side chains of P₃ Val and P₁Leu are closely packed against each other and have substantialhydrophobic contacts with the enzyme (FIG. 8), especially P₃ interactswith Tyr⁷¹ and Phe¹⁰⁸. In the beta-secretase site of native APP, the P₂and P₁ residues are Lys and Met respectively. Swedish mutant APP has Asnand Leu in these positions respectively, resulting in a 60-fold increaseof K_(cat)/K_(m) over that for native APP and an early onset of ADdescribed by Mullan, M., et al. [Nat. Genet. 2, 340 (1992)]. The currentstructure suggests that inhibitor P₂ Lys would place its positivelycharge in an unfavorable interaction with Arg²³⁵ with a loss of hydrogenbond to Arg²³⁵, while P₁ Met would have less favorable contact withmemapsin 2 than does leucine in this site (FIG. 9). No close contactwith memapsin 2 was seen for P₁′ Ala and an aspartic at this position,as in APP, may be accommodated by interacting with Arg²²⁸.

The direction of inhibitor chain turns at P2′ and leads P₃′ and P₄′toward the protein surface (FIG. 9). As a result, the side-chainposition of P₂′ Ala deviates from the regular extended conformation. Theside chains of P₃′ Glu and P₄′ Phe are both pointed toward molecularsurface with little significant interaction with the protease (FIG. 9).The relatively high B-factors (58.2 Å² for Glu and 75.6 Å² for Phe) andless well-defined electron density suggests that these two residues arerelatively mobile, in contrast to the defined structure of the S₃′ andS₄′ subsites in renin-inhibitor (CH-66) complex (Dealwis et al., 1994).The topologically equivalent region of these renin subsites (residues292-297 in pepsin numbering) is deleted in memapsin 2. Theseobservations suggest that the conformation of three C-terminal residuesof OM99-2 may be a functional feature of memapsin 2, possibly a way tolead a long protein substrate out of the active-site cleft.

Example 10 Using the Crystal Structure to Design Inhibitors

Pharmaceutically acceptable inhibitor drugs normally post a size limitunder 800 daltons. In the case of memapsin 2 inhibitors, thisrequirement may even be more stringent due to the need for the drugs topenetrate the blood-brain barrier [Kearney and Aweeka, (1999)]. In thecurrent model, well defined subsite structures spending P4 to P₂′provide sufficient template areas for rational design of such drugs. Thespacial relationships of individual inhibitor side chain with thecorresponding subsite of the enzyme as revealed in this crystalstructure permits the design of new inhibitor structures in each ofthese positions. It is also possible to incorporate the uniqueconformation of subsites P₂′, P₃′ and P₄′ into the selectivity ofmemapsin 2 inhibitors. The examples of inhibitor design based on thecurrent crystal structure are given below.

Example A

Since the side chains of P₃ Val and P₁ Leu are packed against each otherand there is no enzyme structure between them, cross-linking these sidechains would increase the binding strength of inhibitor to memaspin 2.This is because when binding to the enzyme, the cross-linked inhibitorswould have less entropy difference between the free and bound forms thantheir non-cross-linked counterparts [Khan, A. R., et al., Biochemistry,37, 16839 (1998)]. Possible structures of the cross-linked side chainsinclude those shown in FIG. 10.

Example B

The same situation exits between the P4 Glu and P2 Asn. The currentcrystal structure shows that these side chains are already hydrogenbonded to each other so the cross linking between them would also derivebinding benefit as described in the Example A. The cross-linkedstructures include those shown in FIG. 11.

Example C

Based on the current crystal structure, the P1′ Ala side chain may beextended to add new hydrophobic, Van der Waals and H-bond interactions.An example of such a design is diagramed in FIG. 12.

Example D

Based on the current crystal structure, the polypeptide backbone in theregion of P1, P2, and P3, and the side chain of P1-Leu can be bridgedinto rings by the addition of two atoms (A and B in FIG. 13). Also, amethyl group can be added to the beta-carbon of the P1-Leu (FIG. 13).

Modifications and variations of the methods and materials describedherein will be obvious to those skilled in the art and are intended tocome within the scope of the appended claims.

1. A memapsin 2 protein consisting of SEQ ID NO:
 40. 2. The memapsin 2protein of claim 1, wherein the memapsin 2 protein is expressed in abacterial cell.
 3. The memapsin 2 protein of claim 1, wherein thememapsin 2 protein is refolded after expression in a bacterial cell. 4.The memapsin 2 protein of claim 3, wherein the memapsin 2 protein isrefolded by dissolution in a urea solution having a urea concentrationof about 8M and at least one reducing agent at a pH of at least about pH8.0.
 5. The memapsin 2 protein of claim 4, wherein the memapsin 2protein is refolded by dilution in an aqueous buffer having a pH of atleast about pH 9, and wherein the pH subsequently is adjusted to aboutpH 8, and then maintained at a temperature of about 4° C. for a periodof time in a range of between about 24 hours and about 48 hours.
 6. Thememapsin 2 of claim 4, wherein the memapsin 2 protein is refolded bydilution in an aqueous buffer having a pH of at least about pH 9 andincluding an oxidized and reduced glutathione, and wherein the ureaconcentration of the diluted solution of memapsin 2 protein decreases toabout 4 M, and wherein the pH is adjusted to about pH
 8. 7. The memapsin2 protein of claim 4, wherein the memapsin 2 protein is refolded bydissolution in urea having a urea concentration of about 8 M and a pH ofabout pH 10, wherein a resulting solution subsequently is diluted in anaqueous buffer having a pH of about pH 9.0, and maintaining the solutionat a temperature of about 4° C. for a period of time in a range ofbetween about 24 hours and about 48 hours.